Human N-type calcium channel isoform and uses thereof

ABSTRACT

The invention pertains to a human N-type calcium channel isoform, hα 1B+SFVG , which is involved in central nervous system signaling, and nucleic acids relating thereto. The present invention also includes fragments and biologically functional variants of the human hα 1B+SFVG  channel. Also included are human N-type calcium channel hα 1B+SFVG  subunit inhibitors which inhibit human N-type calcium channel hα 1B+SFVG  subunit activity by inhibiting the expression or function of human N-type calcium channel hα 1B+SFVG  subunit. The invention further relates to methods of using such nucleic acids, polypeptides, and inhibitors in the treatment and/or diagnosis of disease, such as in methods for treating stroke, pain, e.g., neuropathic pain, and traumatic brain injury.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/268,163, filed Mar. 12, 1999, now U.S. Pat. No. 6,353,091 and claimsthe benefit under 35 U.S.C. § 119(e) of U.S. provisional applicationSer. No. 60/077,901, filed Mar. 13, 1998, the disclosures of which areincorporated by reference herein.

FIELD OF THE INVENTION

The invention pertains to human N-type calcium channel α_(1B) subunitisoforms.

BACKGROUND OF THE INVENTION

Voltage gated calcium channels, also known as voltage dependent calciumchannels (VDCCs) are multisubunit membrane spanning proteins whichpermit controlled calcium influx from an extracellular environment intothe interior of a cell. Several types of voltage gated calcium channelhave been described in different tissues, including N-type, P/Q-type,L-type and T-type channels. A voltage gated calcium channel permitsentry into the cell of calcium upon depolarization of the membrane ofthe cell, which is a lessening of the difference in electrical potentialbetween the outside and the inside of the cell.

A voltage gated calcium channel contains several proteins, including all(α₁, α₂, β, and γ subunits. Subtypes of the calcium channel subunitsalso are known. For instance, α₁ subtypes include α_(1A), α_(1B),α_(1C), α_(1D), α_(1E) and α_(1S). Each subunit may have one or moreisoforms which result from alternative splicing of RNA in the formationof a completed messenger RNA which encodes the subunit. For example, atleast four isoforms of the rat N-type α_(1B) subunit are known (see,e.g., Lin et al., Neuron 18:153–166, 1997).

Isoforms of calcium channel α₁ subunits may be expressed differently indifferent tissues (see, e.g., Lin et al., 1997). Differential expressionof subunits isoforms raises the possibility of developing therapeuticswhich are specific for distinct isoforms of the α₁, subunits, therebylessening side effects resulting from the use of therapeutics which areeffective for more than one calcium channel isoform. Two isoforms of thehuman N-type calcium channel α_(1B) subunit were published by Williamset al in 1992 (Science 257:389–395). Given the existence of severaladditional rat isoforms in a highly conserved gene family, it issurprising that additional human isoforms of the N-type calcium channelα_(1B) subunit have not been discovered. Such isoforms would be usefulfor developing isoform-specific therapeutics.

SUMMARY OF THE INVENTION

The invention provides isolated nucleic acid molecules, unique fragmentsof those molecules, expression vectors containing the foregoing, andhost cells transfected with those molecules. The invention also providesisolated polypeptides and inhibitors of the foregoing nucleic acids andpolypeptides which reduce voltage-gated calcium influx. The foregoingcan be used in the diagnosis or treatment of conditions characterized byincreased or decreased human N-type calcium channel hα_(1B+SFVG) subunitactivity and can be used in methods in which it is therapeuticallyuseful to increase or decrease human N-type calcium channel hα_(1B+SFVG)subunit activity such as treatments for stroke, pain (e.g., neuropathicpain), traumatic brain injury and conditions characterized by increasedor decreased voltage regulated calcium influx. Here, we present theidentification of a novel human N-type calcium channel α_(1B) subunit,hα_(1B+SFVG), which plays a role in voltage-gated calcium influx.

It was discovered that a brain α_(1B) calcium channel subunit isoform(splice variant) contains a four amino acid insert relative to publishedhuman α_(1B) calcium channel isoforms (SEQ ID NO:5 [GenBank accessionnumber M94172], SEQ ID NO:7 [GenBank accession number M94173]).Surprisingly, this insert, SFVG (SEQ ID NO:2, encoded by SEQ ID NO:1),is similar but not identical to an insert found in a rat α_(1B) channel(GenBank accession number M92905). A significant proportion of the humanN-type calcium channel α_(1B) subunit mRNA in brain was found to be thehα_(1B+SFVG) sub-type; given the abundance of its expression theisolation of this sub-type so long after the identification of otherα_(1B) isoforms is unexpected. The SFVG-containing human N-type calciumchannel hα_(1B+SFVG) subunit also lacks an amino acid sequence, ET,which is present in published human N-type calcium channel hα_(1B+SFVG)subunit isoforms (amino acids 1557–1558 of SEQ ID NOs:5 and 7).

The invention involves in one aspect an isolated human N-type calciumchannel α_(1B) subunit polypeptide which includes the amino acidsequence of SEQ ID NO: 2 (an hα_(1B+SFVG) polypeptide). In oneembodiment, the polypeptide comprises the amino acid sequence of SEQ IDNO:4, and preferably consists of the amino acid sequence of SEQ ID NO:4.In another embodiment the hα_(1B+SFVG) calcium channel polypeptide is afragment or variant of the foregoing polypeptides, wherein the fragmentor variant includes the amino acid sequence of SEQ ID NO:2 or additions,deletions or substitutions thereof which confer the same function as SEQID NO: 2. Preferred variants include those having additions,substitutions or deletions relative to the human N-type calcium channelhα_(1B+SFVG) SFVG subunit polypeptide sequence disclosed herein,particularly those variants which retain one or more of the activitiesof the human N-type calcium channel hα_(1B+SFVG) subunit, includingsubunits with or without the ET exon sequence.

According to another aspect of the invention, an isolated nucleic acidmolecule which encodes any of the foregoing human N-type calcium channelhα_(1B+SFVG) subunit polypeptide is provided. In certain embodiments,the nucleic acid molecule includes SEQ ID NO:1. In one preferredembodiment, the human N-type calcium channel hα_(1B+SFVG) subunitpolypeptides is encoded by a nucleic acid molecule which comprises thenucleotide sequence of SEQ ID NO:3 (Williams et al. sequence +SFVG,−ET), and which preferably consists of the nucleotide sequence of SEQ IDNO:3. In another embodiment the nucleic acid is an allele of the nucleicacid sequence of SEQ ID NO:3.

In another aspect the invention is an expression vector comprising thehuman N-type calcium channel hα_(1B+SFVG) subunit nucleic acid moleculeoperably linked to a promoter. Also included within the invention is ahost cell transformed or transfected with the expression vector.

According to another aspect of the invention, an agent which selectivelybinds the human N-type calcium channel hα_(1B+SFVG) subunit polypeptideor a nucleic acid that encodes the human N-type calcium channelhα_(1B+SFVG) subunit polypeptide is provided. By “selectively binds” itis meant that the agent binds the human N-type calcium channelhα_(1B+SFVG) subunit polypeptide or nucleic acid, or any fragmentthereof which retains the amino acids of SEQ ID NO:2 or the nucleotidesof SEQ ID NO:1, to a greater extent than the agent binds other humanN-type calcium channel α_(1B) subunit isoforms, and preferably does notbind other human N-type calcium channel α_(1B) subunit isoforms. In oneembodiment, the agent is a polypeptide which binds selectively to thehuman N-type calcium channel hα_(1B+SFVG) subunit polypeptide. Thepolypeptide can be a monoclonal antibody, a polyclonal antibody, or anantibody fragment selected from the group consisting of a Fab fragment,a F(ab)₂ fragment and a fragment including a CDR3 region. In anotherembodiment, the agent is an antisense nucleic acid which selectivelybinds to a nucleic acid encoding the human N-type calcium channelhα_(1B+SFVG) subunit polypeptide. Preferably the foregoing agents areinhibitors (antagonists) or agonists of the calcium channel activity ofthe human N-type calcium channel hα_(1B+SFVG) subunit polypeptide.

According to another aspect of the inventions, a dominant negative humanN-type calcium channel hα_(1B+SFVG) subunit polypeptide is provided. Thedominant negative polypeptide is an inhibitor of the function of thecalcium channel.

The invention also provides compositions including any of the foregoingpolypeptides, nucleic acids or agents in combination with apharmaceutically acceptable carrier.

In another aspect of the invention a method for inhibiting human N-typecalcium channel hα_(1B+SFVG) subunit activity in a mammalian cell isprovided. The method involves the step of contacting the mammalian cellwith an amount of a human N-type calcium channel hα_(1B+SFVG) subunitinhibitor effective to inhibit calcium influx in the mammalian cell.Preferably the inhibitor is selected from the group consisting of apeptide or an antibody which selectively binds the human N-type calciumchannel hα_(1B+SFVG) subunit polypeptide, an antisense nucleic acidwhich binds a nucleic acid encoding human N-type calcium channelhα_(1B+SFVG) subunit polypeptide and a dominant negative human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide.

According to still another aspect the invention, a method for treating asubject having a stroke, pain (e.g., neuropathic pain), or traumaticbrain injury is provided. The method involves the step of administeringto a subject in need of such treatment an inhibitor of the human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide in an amount effectiveto inhibit voltage regulated calcium influx. In another embodiment ofthe foregoing methods, the inhibitor is administered prophylactically toa subject at risk of having a stroke.

The human N-type calcium channel hα_(1B+SFVG) subunit polypeptides andnucleic acids which encode such polypeptides are useful for increasingthe amount of human N-type calcium channel hα_(1B+SFVG) subunitpolypeptides in a cell. Increasing the amount of human N-type calciumchannel hα_(1B+SFVG) subunit polypeptides in a cell results in increasedvoltage regulated calcium influx. This is useful where it is desired toincrease the amount of voltage regulated calcium influx which ismediated by a human N-type calcium channel.

Thus according to another aspect of the invention, a method forincreasing human N-type calcium channel hα_(1B+SFVG) subunit expressionin a cell is provided. The method involves the step of contacting thecell with a molecule selected from the group consisting of a humanN-type calcium channel hα_(1B+SFVG) subunit nucleic acid and a humanN-type calcium channel hα_(1B+SFVG) subunit polypeptide in an amounteffective to increase voltage regulated calcium influx in the cell. Incertain embodiments, the cell is contacted with one or more human N-typecalcium channel non-hα_(1B+SFVG) subunits, such as a β subunit, ornucleic acids encoding such non-hα_(1B+SFVG) subunits.

According to another aspect of the invention, a method for increasingcalcium channel voltage regulated calcium influx in a subject isprovided. The method involves the step of administering to a subject inneed of such treatment a molecule selected from the group consisting ofa human N-type calcium channel hα_(1B+SFVG) subunit nucleic acid and ahuman N-type calcium channel hα_(1B+SFVG) subunit polypeptide in anamount effective to increase voltage regulated calcium influx in thesubject.

According to a further aspect of the invention, a method for identifyinglead compounds for a pharmacological agent useful in the treatment ofdisease associated with increased or decreased voltage regulated calciuminflux mediated by a human N-type calcium channel is provided. A cell orother membrane-encapsulated space comprising a human N-type calciumchannel hα_(1B+SFVG) subunit polypeptide is provided. The cell or othermembrane-encapsulated space preferably is loaded with acalcium-sensitive compound which is detectable in the presence ofcalcium. The cell or other membrane-encapsulated space is contacted witha candidate pharmacological agent under conditions which, in the absenceof the candidate pharmacological agent, cause a first amount of voltageregulated calcium influx into the cell or other membrane-encapsulatedspace. A test amount of voltage regulated calcium influx then isdetermined. For example, in a preferred embodiment, fluorescence of acalcium-sensitive compound then is detected as a measure of the voltageregulated calcium influx. If the test amount of voltage regulatedcalcium influx is less than the first amount, then the candidatepharmacological agent is a lead compound for a pharmacological agentwhich reduces voltage regulated calcium influx. If the test amount ofvoltage regulated calcium influx is greater than the first amount, thenthe candidate pharmacological agent is a lead compound for apharmacological agent which increases voltage regulated calcium influx.

In another aspect of the invention, methods for identifying compoundswhich selectively or preferentially bind a human N-type calcium channelhα_(1B+SFVG) subunit isoform are provided. In one embodiment, the methodincludes providing a first cell or membrane encapsulated space whichexpresses a human N-type calcium channel hα_(1B+SFVG) subunit isoform,and providing a second cell or membrane encapsulated space whichexpresses a human N-type calcium channel non-hα_(1B+SFVG) subunitisoform, wherein the second cell or membrane encapsulated space isidentical to the first cell except for the α_(1B) isoform expressed. Thefirst cell or membrane encapsulated space and the second cell ormembrane encapsulated space are contacted with a compound, and thebinding of the compound to the first cell or membrane encapsulated spaceand the second cell or membrane encapsulated space is determined. Acompound which binds the first cell or membrane encapsulated space butdoes not bind the second cell or membrane encapsulated space is acompound which selectively binds the human N-type calcium channelhα_(1B+SFVG) subunit isoform. A compound which binds the first cell ormembrane encapsulated space in an amount greater than the compound bindsthe second cell or membrane encapsulated space is a compound whichpreferentially binds the human N-type calcium channel hα_(1B+SFVG)subunit isoform. In another embodiment of the method, a human N-typecalcium channel hα_(1B+SFVG) subunit isoform polypeptide or nucleic acidand a human N-type calcium channel non-hα_(1B+SFVG) subunit isoformpolypeptide or nucleic acid are provided and contacted with a compound.The binding of the compound to the human N-type calcium channelhα_(1B+SFVG) subunit isoform polypeptide or nucleic acid and the humanN-type calcium channel non-hα_(1B+SFVG) subunit isoform polypeptide ornucleic acid then is determined. A compound which binds the human N-typecalcium channel hα_(1B+SFVG) subunit isoform polypeptide or nucleic acidbut does not bind the human N-type calcium channel non-hα_(1B+SFVG)subunit isoform polypeptide or nucleic acid is a compound whichselectively binds the human N-type calcium channel hα_(1B+SFVG) subunitisoform polypeptide or nucleic acid. A compound which binds the humanN-type calcium channel hα_(1B+SFVG) subunit isoform polypeptide ornucleic acid in an amount greater than the human N-type calcium channelnon-hα_(1B+SFVG) subunit isoform polypeptide or nucleic acid is acompound which preferentially binds the human N-type calcium channelhα_(1B+SFVG) subunit isoform polypeptide or nucleic acid. Also includedin the invention are compounds identified using the foregoing methods.

According to another aspect of the invention, a method for selectivelytreating a subject having a condition characterized by aberrant brainneuronal calcium current is provided. The method includes the step ofadministering to a subject in need of such treatment a pharmacologicalagent which is selective for a human N-type calcium channel hα_(1B+SFVG)subunit, in an amount effective to normalize the aberrant neuronalcalcium current. Aberrant means a level of calcium current (calciuminflux) which is outside of a normal range as understood in the medicalarts. Normalize means that the calcium current is brought within thenormal range.

Also presented herein is an identification of characteristics of certaincalcium channel subunit isoforms with respect to voltage-dependentactivation. It has been discovered, surprisingly, that the presence orabsence of an exon comprising the amino acids ET is important for thekinetics of channel activation. Thus, in still other aspects of theinvention, a variety of novel assays, screens, recombinant products,model systems (such as animal models) and methods are provided whichutilize the unexpected different activation functions between and amongthe calcium channel subunit isoforms for the identification of novelagents, treatments, etc. useful in the modulation of conditions whicharise from or manifest differences in action potential neurotransmitterrelease, voltage-dependent calcium channel activation, and so on. Forexample, methods for the identification of agents which alter activationpotential dependent neurotransmitter release are provided. The methodsinclude selecting an agent which binds a calcium channel isoform havingor lacking a IVS3-S4 ET exon as described herein, and determiningcalcium channel activation or activation potential dependentneurotransmitter release in the presence and the absence of the agent.In some embodiments, candidate compounds may be screened by suchmethods. The methods also can include measurement of these parameters inother calcium channel subunits which manifest such differences inactivation kinetics, including subunits in which an NP exon is added oris substituted for the ET exon.

Use of the foregoing compositions in the preparation of a medicament,and particularly in the preparation of a medicament for the treatment ofstroke, pain (e.g., neuropathic pain), traumatic brain injury, or acondition which results from excessive or insufficient voltage regulatedcalcium influx, is provided.

These and other aspects of the invention are described in greater detailbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that the presence of ET in domain IVS3-S4 of α_(1B) slowsthe rate of N-type Ca channel activation.

FIG. 2 shows the impact of alternative splicing in the S3-S4 linkers ofthe α_(1B) subunit on action potential-dependent Ca influx in a modelneuron.

FIG. 3 shows the results of a functional analysis of site-directedmutagenesis of ET splice site in domain IVS3-S4 of the α_(1B) subunit.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence of the human N-type calciumchannel hα_(1B+SFVG) subunit cDNA IIIS3-S4 “SFVG” site.

SEQ ID NO:2 is the amino acid sequence of the human N-type calciumchannel hα_(1B+SFVG) subunit polypeptide IIIS3-S4 “SFVG” site.

SEQ ID NO:3 is the nucleotide sequence of the human N-type calciumchannel hα_(1B+SFVG) subunit cDNA.

SEQ ID NO:4 is the amino acid sequence of the human N-type calciumchannel hα_(1B+SFVG) subunit polypeptide.

SEQ ID NO:5 is the nucleotide sequence of the coding region of a humanα_(1B) calcium channel which lacks the IIIS3-S4 “SFVG” site (Prior art,GenBank accession number M94172).

SEQ ID NO:6 is the amino acid sequence of a human α_(1B) calcium channelwhich lacks the IIIS3-S4 “SFVG” site (Prior art, GenBank accessionnumber M94172).

SEQ ID NO:7 is the nucleotide sequence of the coding region of a humanα_(1B) calcium channel which lacks the IIIS3-S4 “SFVG” site (Prior art,GenBank accession number M94173).

SEQ ID NO:8 is the amino acid sequence of a human α_(1B) calcium channelwhich lacks the IIIS3-S4 “SFVG” site (Prior art, GenBank accessionnumber M94173).

SEQ ID NO:9 is the nucleotide sequence of the coding region of a ratα_(1B) calcium channel which contains a SFMG site (Prior art, GenBankaccession number M92905).

SEQ ID NO:10 is the amino acid sequence of a rat α_(1B) calcium channelwhich contains a SFMG site (Prior art, GenBank accession number M92905).

SEQ ID NO:11 is the amino acid sequence of an ω-conotoxin peptide fromC. geographus.

SEQ ID NO:12 is the amino acid sequence of ω-conotoxin peptide from C.magus.

SEQ ID NOS:13–28 are primers for PCR and/or sequencing.

DETAILED DESCRIPTION OF THE INVENTION

The present invention in one aspect involves the identification of acDNA encoding a novel human isoform of the N-type calcium channel,referred to herein as the human N-type calcium channel hα_(1B+SFVG)subunit. As used herein, hα_(1B+SFVG) refers to any human N-type calciumchannel α_(1B) subunit clone that contains the SFVG sequence set forthin SEQ ID NO:2. The nucleotide sequence of the human N-type calciumchannel hα_(1B+SFVG) subunit insert, the IIIS3-S4 “SFVG” site, ispresented as SEQ ID NO:1, and the amino acid sequence of the humanN-type calcium channel hα_(1B+SFVG) subunit insert, the IIIS3-S4 “SFVG”site, is presented as SEQ ID NO:2. The 12 nucleotides of SEQ ID NO:1 areinserted immediately following nt 3855 in the coding sequence of thehuman N-type calcium channel α_(1B) subunit in the codon encoding aminoacid 1237, such that the four amino acids of SEQ ID NO:2 are inserted inthe polypeptide after amino acid 1237 (see SEQ ID NO:3). The closelyrelated human N-type calcium channel α_(1B-b) subunit, which does notcontain the IIIS3-S4 “SFVG” site, was deposited in GenBank underaccession numbers M94172 and M94173 (SEQ ID NOs:5–8). A related ratN-type calcium channel α_(1B) subunit was deposited in GenBank underaccession number M92905 (SEQ ID NOs:9 and 10). Surprisingly, the aminoacid sequence of the human N-type calcium channel hα_(1B+SFVG) subunitdiffers from the rat amino acid sequence in the SFVG site, whichsequence is located in an area of the molecule in which the human andrat amino acid sequences are otherwise 100% identical. This speciesdifference in the very highly conserved protein domain of the humanN-type calcium channel hα_(1B+SFVG) subunit is entirely unexpected, andpermits the screening of compounds which selectively bind to and/ormodulate the human N-type calcium channel hα_(1B+SFVG) subunit. Becausethe present human N-type calcium channel hα_(1B+SFVG) subunit is asplice variant of other human N-type calcium channel α_(1B) subunits, itis apparent that the invention is meant to embrace human N-type calciumchannel α_(1B) subunit variants which vary by alternative splicing ofsequences other than the SFVG (SEQ ID NO:2) insert. For example, theinvention embraces polypeptides which contain or do not contain an Alaresidue immediately following amino acid position 414 of SEQ ID NO:3, ora Glu-Thr insert (ET in single letter code) at amino acid positions1557–1558 (see, e.g., SEQ ID NO:6), as well as nucleic acid moleculesencoding such splice variant polypeptides. As shown in the Examples, thehα_(1B+SFVG) subunit is a significant portion of the α_(1B) calciumchannel expressed in human brain, and is differentially distributed indifferent parts of the brain. This opens the possibility for theselective treatment of disorders which involve those parts of the brain.

The invention involves in one aspect human N-type calcium channelhα_(1B+SFVG) subunit nucleic acids and polypeptides, as well astherapeutics relating thereto. The invention also embraces isolatedfunctionally equivalent variants, useful analogs and fragments of theforegoing nucleic acids and polypeptides; complements of the foregoingnucleic acids; and molecules which selectively bind the foregoingnucleic acids and polypeptides.

The human N-type calcium channel hα_(1B+SFVG) subunit nucleic acids andpolypeptides of the invention are isolated. The term “isolated”, as usedherein in reference to a nucleic acid molecule, means a nucleic acidsequence: (i) amplified in vitro by, for example, polymerase chainreaction (PCR); (ii) synthesized by, for example, chemical synthesis;(iii) recombinantly produced by cloning; or (iv) purified, as bycleavage and electrophoretic or chromatographic separation. The term“isolated”, as used herein in reference to a polypeptide, means apolypeptide encoded by an isolated nucleic acid sequence, as well aspolypeptides synthesized by, for example, chemical synthetic methods,and polypeptides separated from biological materials, and then purified,using conventional protein analytical or preparatory procedures, to anextent that permits them to be used according to the methods describedherein.

As used herein a human N-type calcium channel hα_(1B+SFVG) subunitnucleic acid refers to an isolated nucleic acid molecule which codes fora human N-type calcium channel hα_(1B+SFVG) subunit. Human N-typecalcium channel hα_(1B+SFVG) subunit nucleic acids are those nucleicacid molecules which code for human N-type calcium channel hα_(1B+SFVG)subunit polypeptides which include the sequence of SEQ ID NO:2. Thenucleic acid molecules include the nucleotide sequence of SEQ ID NO:1and nucleotide sequences which differ from the sequence of SEQ ID NO:1in codon sequence due to the degeneracy of the genetic code. The humanN-type calcium channel hα_(1B+SFVG) subunit nucleic acids of theinvention also include alleles of the foregoing nucleic acids, as wellas fragments of the foregoing nucleic acids, provided that the allele orfragment encodes the amino acid sequence of SEQ ID NO:2. Such fragmentscan be used, for example, as probes in hybridization assays and asprimers in a polymerase chain reaction (PCR). Preferred human N-typecalcium channel hα_(1B+SFVG) subunit nucleic acids include the nucleicacid sequence of SEQ ID NO: 1. Complements of the foregoing nucleicacids also are embraced by the invention.

As used herein “human N-type calcium channel hα_(1B+SFVG) subunitactivity” refers to an ability of a molecule to modulate voltageregulated calcium influx. A molecule which inhibits human N-type calciumchannel hα_(1B+SFVG) subunit activity (an antagonist) is one whichinhibits voltage regulated calcium influx via this calcium channel and amolecule which increases human N-type calcium channel hα_(1B+SFVG)subunit activity (an agonist) is one which increases voltage regulatedcalcium influx via this calcium channel. Changes in human N-type calciumchannel hα_(1B+SFVG) subunit activity can be measured by changes involtage regulated calcium influx by in vitro assays such as thosedisclosed herein, including patch-clamp assays and assays employingcalcium sensitive fluorescent compounds such as fura-2.

Alleles of the human N-type calcium channel hα_(1B+SFVG) subunit nucleicacids of the invention can be identified by conventional techniques. Forexample, alleles of human N-type calcium channel hα_(1B+SFVG) subunitcan be isolated by hybridizing a probe which includes SEQ ID NO:1 understringent conditions with a cDNA library and selecting positive clones.Thus, an aspect of the invention is those nucleic acid sequences whichcode for human N-type calcium channel hα_(1B+SFVG) subunit polypeptidesand which hybridize to a nucleic acid molecule consisting of SEQ ID NO:1under stringent conditions. The term “stringent conditions” as usedherein refers to parameters with which the art is familiar. Nucleic acidhybridization parameters may be found in references which compile suchmethods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, etal., eds., Second Edition, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Morespecifically, stringent conditions, as used herein, refers, for example,to hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02%Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mMNaH₂PO₄(pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodium chloride/0.015Msodium citrate, pH7; SDS is sodium dodecyl sulphate; and EDTA isethylenediaminetetracetic acid. After hybridization, the membrane uponwhich the DNA is transferred is washed at 2×SSC at room temperature andthen at 0.1×SSC/0.1×SDS at temperatures up to 65° C.

There are other conditions, reagents, and so forth which can be used,which result in a similar degree of stringency. The skilled artisan willbe familiar with such conditions, and thus they are not given here. Itwill be understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof alleles of human N-type calcium channel hα_(1B+SFVG) subunit nucleicacids of the invention. The skilled artisan also is familiar with themethodology for screening cells and libraries for expression of suchmolecules which then are routinely isolated, followed by isolation ofthe pertinent nucleic acid molecule and sequencing.

In screening for human N-type calcium channel hα_(1B+SFVG) subunitnucleic acids, a Southern blot may be performed using the foregoingstringent conditions, together with a radioactive probe. After washingthe membrane to which the DNA is finally transferred, the membrane canbe placed against X-ray film to detect the radioactive signal.

The human N-type calcium channel hα_(1B+SFVG) subunit nucleic acids ofthe invention also include degenerate nucleic acids which includealternative codons to those present in the native materials. Forexample, serine residues are encoded by the codons TCA, AGT, TCC, TCG,TCT and AGC. Each of the six codons is equivalent for the purposes ofencoding a serine residue. Thus, it will be apparent to one of ordinaryskill in the art that any of the serine-encoding nucleotide triplets maybe employed to direct the protein synthesis apparatus, in vitro or invivo, to incorporate a serine residue into an elongating human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide. Similarly, nucleotidesequence triplets which encode other amino acid residues include, butare not limited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC,CGG, CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT(threonine codons); AAC and AAT (asparagine codons); and ATA, ATC andATT (isoleucine codons). Other amino acid residues may be encodedsimilarly by multiple nucleotide sequences. Thus, the invention embracesdegenerate nucleic acids that differ from the biologically isolatednucleic acids in codon sequence due to the degeneracy of the geneticcode.

The invention also provides isolated fragments of SEQ ID NO:3 whichinclude the nucleotide sequence of SEQ ID NO:1. The fragments can beused as probes in Southern blot assays to identify such nucleic acids,or can be used in amplification assays such as those employing PCR.Smaller fragments are those comprising 12, 13, 14, 15, 16, 17, 18, 20,22, 25, 30, 40, 50, or 75 nucleotides, and every integer therebetweenand are useful e.g. as primers for nucleic acid amplificationprocedures. As known to those skilled in the art, larger probes such as200, 250, 300, 400 or more nucleotides are preferred for certain usessuch as Southern blots, while smaller fragments will be preferred foruses such as PCR. Fragments also can be used to produce fusion proteinsfor generating antibodies or determining binding of the polypeptidefragments. Likewise, fragments can be employed to produce non-fusedfragments of the human N-type calcium channel hα_(1B+SFVG) subunitpolypeptides, useful, for example, in the preparation of antibodies, inimmunoassays, and the like. The foregoing nucleic acid fragments furthercan be used as antisense molecules to inhibit the expression of humanN-type calcium channel hα_(1B+SFVG) subunit nucleic acids andpolypeptides, particularly for therapeutic purposes as described ingreater detail below.

The invention also includes functionally equivalent variants of thehuman N-type calcium channel hα_(1B+SFVG) subunit, which include variantnucleic acids and polypeptide which retain one or more of the functionalproperties of the human N-type calcium channel hα_(1B+SFVG) subunit, butalways including SEQ ID NO:2. For example, variants include a fusionprotein which includes the extracellular and transmembrane domains ofthe human N-type calcium channel hα_(1B+SFVG) subunit (including SEQ IDNO:2), which retains the ability to bind ligand and/or transduce avoltage gated calcium current. Still other functionally equivalentvariants include variants of SEQ ID NO: 2 which retain functions ofsubunit including SEQ ID NO: 2. Functionally equivalent variants alsoinclude a human N-type calcium channel hα_(1B+SFVG) subunit which hashad a portion of the extracellular domain (but not SEQ ID NO:2) removedor replaced by a similar domain from another calcium channel α₁ subunit(e.g. a “domain-swapping” variant). Other functionally equivalentvariants will be known to one of ordinary skill in the art, as willmethods for preparing such variants. The activity of a functionallyequivalent variant can be determined using the methods provided herein,in Lin et al., Neuron 18:153–166, 1997, and in U.S. Pat. No. 5,429,921.Such variants are useful, inter alia, in assays for identification ofcompounds which bind and/or regulate the calcium influx function of thehuman N-type calcium channel hα_(1B+SFVG) subunit, and for determiningthe portions of the human N-type calcium channel hα_(1B+SFVG) subunitwhich are required for calcium influx activity.

Variants which are non-functional also can be prepared as describedabove. Such variants are useful, for example, as negative controls inexperiments testing subunit activity, and as inhibition of N-typecalcium channel activity.

A human N-type calcium channel hα_(1B+SFVG) subunit nucleic acid, in oneembodiment, is operably linked to a gene expression sequence whichdirects the expression of the human N-type calcium channel hα_(1B+SFVG)subunit nucleic acid within a eukaryotic or prokaryotic cell. The “geneexpression sequence” is any regulatory nucleotide sequence, such as apromoter sequence or promoter-enhancer combination, which facilitatesthe efficient transcription and translation of the human N-type calciumchannel hα_(1B+SFVG) subunit nucleic acid to which it is operablylinked. The gene expression sequence may, for example, be a mammalian orviral promoter, such as a constitutive or inducible promoter.Constitutive mammalian promoters include, but are not limited to, thepromoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, β-actinpromoter and other constitutive promoters. Exemplary viral promoterswhich function constitutively in eukaryotic cells include, for example,promoters from the simian virus, papilloma virus, adenovirus, humanimmunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, thelong terminal repeats (LTR) of Moloney murine leukemia virus and otherretroviruses, and the thymidine kinase promoter of herpes simplex virus.Other constitutive promoters are known to those of ordinary skill in theart. The promoters useful as gene expression sequences of the inventionalso include inducible promoters. Inducible promoters are expressed inthe presence of an inducing agent. For example, the metallothioneinpromoter is induced to promote transcription and translation in thepresence of certain metal ions. Other inducible promoters are known tothose of ordinary skill in the art.

In general, the gene expression sequence shall include, as necessary, 5′non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription and translation, respectively, such as aTATA box, capping sequence, CAAT sequence, and the like. Especially,such 5′ non-transcribing sequences will include a promoter region whichincludes a promoter sequence for transcriptional control of the operablyjoined human N-type calcium channel hα_(1B+SFVG) subunit nucleic acid.The gene expression sequences optionally includes enhancer sequences orupstream activator sequences as desired.

The human N-type calcium channel hα_(1B+SFVG) subunit nucleic acidsequence and the gene expression sequence are said to be “operablylinked” when they are covalently linked in such a way as to place thetranscription and/or translation of the human N-type calcium channelhα_(1B+SFVG) subunit coding sequence under the influence or control ofthe gene expression sequence. If it is desired that the human N-typecalcium channel hα_(1B+SFVG) subunit sequence be translated into afunctional protein, two DNA sequences are said to be operably linked ifinduction of a promoter in the 5′ gene expression sequence results inthe transcription of the human N-type calcium channel hα_(1B+SFVG)subunit sequence and if the nature of the linkage between the two DNAsequences does not (1) result in the introduction of a frame-shiftmutation, (2) interfere with the ability of the promoter region todirect the transcription of the human N-type calcium channelhα_(1B+SFVG) subunit sequence, or (3) interfere with the ability of thecorresponding RNA transcript to be translated into a protein. Thus, agene expression sequence would be operably linked to a human N-typecalcium channel hα_(1B+SFVG) subunit nucleic acid sequence if the geneexpression sequence were capable of effecting transcription of thathuman N-type calcium channel hα_(1B+SFVG) subunit nucleic acid sequencesuch that the resulting transcript might be translated into the desiredprotein or polypeptide.

The human N-type calcium channel hα_(1B+SFVG) subunit nucleic acid andthe human N-type calcium channel hα_(1B+SFVG) subunit polypeptide(including the human N-type calcium channel hα_(1B+SFVG) subunitinhibitors described below) of the invention can be delivered to theeukaryotic or prokaryotic cell alone or in association with a vector. Inits broadest sense, a “vector” is any vehicle capable of facilitating:(1) delivery of a human N-type calcium channel hα_(1B+SFVG) subunitnucleic acid or polypeptide to a target cell or (2) uptake of a humanN-type calcium channel hα_(1B+SFVG) subunit nucleic acid or polypeptideby a target cell. Preferably, the vectors transport the human N-typecalcium channel hα_(1B+SFVG) subunit nucleic acid or polypeptide intothe target cell with reduced degradation relative to the extent ofdegradation that would result in the absence of the vector. Optionally,a “targeting ligand” can be attached to the vector to selectivelydeliver the vector to a cell which expresses on its surface the cognatereceptor (e.g. a receptor, an antigen recognized by an antibody) for thetargeting ligand. In this manner, the vector (containing a human N-typecalcium channel hα_(1B+SFVG) subunit nucleic acid or a human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide) can be selectivelydelivered to a specific cell. In general, the vectors useful in theinvention are divided into two classes: biological vectors andchemical/physical vectors. Biological vectors are more useful fordelivery/uptake of human N-type calcium channel hα_(1B+SFVG) subunitnucleic acids to/by a target cell. Chemical/physical vectors are moreuseful for delivery/uptake of human N-type calcium channel hα_(1B+SFVG)subunit nucleic acids or human N-type calcium channel hα_(1B+SFVG)subunit proteins to/by a target cell.

Biological vectors include, but are not limited to, plasmids, phagemids,viruses, other vehicles derived from viral or bacterial sources thathave been manipulated by the insertion or incorporation of the nucleicacid sequences of the invention, and free nucleic acid fragments whichcan be attached to the nucleic acid sequences of the invention. Viralvectors are a preferred type of biological vector and include, but arenot limited to, nucleic acid sequences from the following viruses:retroviruses, such as Moloney murine leukemia virus; Harvey murinesarcoma virus; murine mammary tumor virus; Rous sarcoma virus;adenovirus; adeno-associated virus; SV40-type viruses; polyoma viruses;Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus;and polio virus. One can readily employ other vectors not named butknown in the art.

Preferred viral vectors are based on non-cytopathic eukaryotic virusesin which non-essential genes have been replaced with the gene ofinterest. Non-cytopathic viruses include retroviruses, the life cycle ofwhich involves reverse transcription of genomic viral RNA into DNA withsubsequent proviral integration into host cellular DNA. In general, theretroviruses are replication-deficient (i.e., capable of directingsynthesis of the desired proteins, but incapable of manufacturing aninfectious particle). Such genetically altered retroviral expressionvectors have general utility for the high-efficiency transduction ofgenes in vivo. Standard protocols for producing replication-deficientretroviruses (including the steps of incorporation of exogenous geneticmaterial into a plasmid, transfection of a packaging cell line withplasmid, production of recombinant retroviruses by the packaging cellline, collection of viral particles from tissue culture media, andinfection of the target cells with viral particles) are provided inKriegler, M., “Gene Transfer and Expression, A Laboratory Manual, ” W.H. Freeman C. O., New York (1990) and Murry, E. J. Ed. “Methods inMolecular Biology,” vol. 7, Humana Press, Inc., Clifton, N.J. (1991).

Another preferred virus for certain applications is the adeno-associatedvirus, a double-stranded DNA virus. The adeno-associated virus can beengineered to be replication-deficient and is capable of infecting awide range of cell types and species. It further has advantages, such asheat and lipid solvent stability; high transduction frequencies in cellsof diverse lineages, including hemopoietic cells; and lack ofsuperinfection inhibition thus allowing multiple series oftransductions. Reportedly, the adeno-associated virus can integrate intohuman cellular DNA in a site-specific manner, thereby minimizing thepossibility of insertional mutagenesis and variability of inserted geneexpression. In addition, wild-type adeno-associated virus infectionshave been followed in tissue culture for greater than 100 passages inthe absence of selective pressure, implying that the adeno-associatedvirus genomic integration is a relatively stable event. Theadeno-associated virus can also function in an extrachromosomal fashion.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA (RNA) encoding a human N-type calcium channelhα_(1B+SFVG) subunit polypeptide or fragment or variant thereof. Thatheterologous DNA (RNA) is placed under operable control oftranscriptional elements to permit the expression of the heterologousDNA in the host cell.

Preferred systems for mRNA expression in mammalian cells are those suchas pRc/CMV (available from Invitrogen, Carlsbad, Calif.) that contain aselectable marker such as a gene that confers G418 resistance (whichfacilitates the selection of stably transfected cell lines) and thehuman cytomegalovirus (CMV) enhancer-promoter sequences. Additionally,suitable for expression in primate or canine cell lines is the pCEP4vector (Invitrogen), which contains an Epstein Barr virus (EBV) originof replication, facilitating the maintenance of plasmid as a multicopyextrachromosomal element. Another expression vector is the pEF-BOSplasmid containing the promoter of polypeptide Elongation Factor 1α,which stimulates efficiently transcription in vitro. The plasmid isdescribed by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), andits use in transfection experiments is disclosed by, for example,Demoulin (Mol. Cell. Biol. 16:4710–4716, 1996). Still another preferredexpression vector is an adenovirus, described by Stratford-Perricaudet,which is defective for E1 and E3 proteins (J. Clin. Invest. 90:626–630,1992).

In addition to the biological vectors, chemical/physical vectors may beused to deliver a human N-type calcium channel hα_(1B+SFVG) subunitnucleic acid or polypeptide to a target cell and facilitate uptakethereby. As used herein, a “chemical/physical vector” refers to anatural or synthetic molecule, other than those derived frombacteriological or viral sources, capable of delivering the isolatedhuman N-type calcium channel hα_(1B+SFVG) subunit nucleic acid orpolypeptide to a cell.

A preferred chemical/physical vector of the invention is a colloidaldispersion system. Colloidal dispersion systems include lipid-basedsystems including oil-in-water emulsions, micelles, mixed micelles, andliposomes. A preferred colloidal system of the invention is a liposome.Liposomes are artificial membrane vesicles which are useful as adelivery vector in vivo or in vitro. It has been shown that largeunilamellar vesicles (LUV), which range in size from 0.2–4.0 μ canencapsulate large macromolecules. RNA, DNA, and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (Fraley, et al., Trends Biochem. Sci., v. 6, p.77 (1981)). In order for a liposome to be an efficient nucleic acidtransfer vector, one or more of the following characteristics should bepresent: (1) encapsulation of the nucleic acid of interest at highefficiency with retention of biological activity; (2) preferential andsubstantial binding to a target cell in comparison to non-target cells;(3) delivery of the aqueous contents of the vesicle to the target cellcytoplasm at high efficiency; and (4) accurate and effective expressionof genetic information.

Liposomes may be targeted to a particular tissue by coupling theliposome to a specific ligand such as a monoclonal antibody, sugar,glycolipid, or protein. Ligands which may be useful for targeting aliposome to a particular cell will depend on the particular cell ortissue type. Additionally when the vector encapsulates a nucleic acid,the vector may be coupled to a nuclear targeting peptide, which willdirect the human N-type calcium channel hα_(1B+SFVG) subunit nucleicacid to the nucleus of the host cell.

Liposomes are commercially available from Gibco BRL, for example, asLIPOFECTIN™ and LIPOFECTACE™, which are formed of cationic lipids suchas N-[1-(2,3dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA)and dimethyl dioctadecylammonium bromide (DDAB). Methods for makingliposomes are well known in the art and have been described in manypublications. Liposomes also have been reviewed by Gregoriadis, G. inTrends in Biotechnology, V. 3, p. 235–241 (1985).

Other exemplary compositions that can be used to facilitate uptake by atarget cell of the human N-type calcium channel hα_(1B+SFVG) subunitnucleic acids include calcium phosphate and other chemical mediators ofintracellular transport, microinjection compositions, electroporationand homologous recombination compositions (e.g., for integrating a humanN-type calcium channel hα_(1B+SFVG) subunit nucleic acid into apreselected location within a target cell chromosome).

The invention also embraces so-called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of the previouslydiscussed coding sequences. Other components may be added, as desired,as long as the previously mentioned sequences, which are required, areincluded.

It will also be recognized that the invention embraces the use of thehuman N-type calcium channel hα_(1B+SFVG) subunit cDNA sequences inexpression vectors, as well as to transfect host cells and cell lines,be these prokaryotic (e.g., E. coli), or eukaryotic (e.g., COS cells,yeast expression systems and recombinant baculovirus expression ininsect cells). Especially useful are mammalian cells such as human, pig,goat, primate, etc. They may be of a wide variety of tissue types, andinclude primary cells and cell lines. Specific examples include neuronalcells including PC12 cells, Xenopus oocytes, bone marrow stem cells andembryonic stem cells. The expression vectors require that the pertinentsequence, i.e., those nucleic acids described supra, be operably linkedto a promoter.

The invention also provides isolated human N-type calcium channelhα_(1B+SFVG) subunit polypeptides which include the amino acid sequenceof SEQ ID NO:2, encoded by the human N-type calcium channel hα_(1B+SFVG)subunit nucleic acids described above. The preferred human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide has the amino acidsequence of SEQ ID NO:4. Human N-type calcium channel hα_(1B+SFVG)subunit polypeptides also embrace alleles, functionally equivalentvariants and analogs (those non-allelic polypeptides which vary in aminoacid sequence from SEQ ID NO:4 by 1, 2, 3, 4, 5, or more amino acids)provided that such polypeptides include the amino acids of SEQ ID NO:2and retain human N-type calcium channel hα_(1B+SFVG) subunit activity,and fragments of SEQ ID NO:4 which include SEQ ID NO:2. Non-functionalvariants also are embraced by the invention; these are useful asantagonists of calcium channel function, as negative controls in assays,and the like. Such alleles, variants, analogs and fragments are useful,for example, alone or as fusion proteins for a variety of purposes suchas to generate antibodies, or as a component of an immunoassay.

Fragments of a polypeptide preferably are those fragments which retain adistinct functional capability of the human N-type calcium channelhα_(1B+SFVG) subunit polypeptide, in particular voltage regulatedcalcium influx. Other functional capabilities which can be retained in afragment of a human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide include interaction with antibodies and interaction withother polypeptides (such as other subunits of the human N-type calciumchannel). Those skilled in the art are well versed in methods forselecting fragments which retain a functional capability of the humanN-type calcium channel hα_(1B+SFVG) subunit. Confirmation of thefunctional capability of the fragment can be carried out by synthesis ofthe fragment and testing of the capability according to standardmethods. For example, to test the voltage regulated calcium influx of ahuman N-type calcium channel hα_(1B+SFVG) subunit fragment, one insertsor expresses the fragment in a cell in which calcium influx can bemeasured. Such methods, which are standard in the art, are describedfurther in the examples.

The invention embraces variants of the human N-type calcium channelhα_(1B+SFVG) subunit polypeptides described above. As used herein, a“variant” of a human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide is a polypeptide which contains one or more modifications tothe primary amino acid sequence of a human N-type calcium channelhα_(1B+SFVG) subunit polypeptide. Modifications which create a humanN-type calcium channel hα_(1B+SFVG) subunit variant can be made to ahuman N-type calcium channel hα_(1B+SFVG) subunit polypeptide for avariety of reasons, including 1) to reduce or eliminate an activity of ahuman N-type calcium channel hα_(1B+SFVG) subunit polypeptide, such asvoltage gated calcium influx; 2) to enhance a property of a human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide, such as proteinstability in an expression system or the stability of protein-proteinbinding; 3) to provide a novel activity or property to a human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide, such as addition of anantigenic epitope or addition of a detectable moiety; or 4) to establishthat an amino acid substitution does or does not affect voltage gatedcalcium influx. Modifications to a human hα_(1B+SFVG) calcium channelpolypeptide are typically made to the nucleic acid which encodes thehuman N-type calcium channel hα_(1B+SFVG) subunit polypeptide, and caninclude deletions, point mutations, truncations, amino acidsubstitutions and additions of amino acids or non-amino acid moieties.Alternatively, modifications can be made directly to the polypeptide,such as by cleavage, addition of a linker molecule, addition of adetectable moiety, such as biotin, addition of a fatty acid, and thelike. Modifications also embrace fusion proteins comprising all or partof the human N-type calcium channel hα_(1B+SFVG) subunit amino acidsequence, but always including SEQ ID NO:2. One of skill in the art willbe familiar with methods for predicting the effect on proteinconformation of a change in protein sequence, and can thus “design” avariant human N-type calcium channel hα_(1B+SFVG) subunit according toknown methods. One example of such a method is described by Dahiyat andMayo in Science 278:82–87, 1997, whereby proteins can be designed denovo. The method can be applied to a known protein to vary a only aportion of the polypeptide sequence. By applying the computationalmethods of Dahiyat and Mayo, specific variants of a cancer associatedantigen polypeptide can be proposed and tested to determine whether thevariant retains a desired conformation.

Variants include human N-type calcium channel hα_(1B+SFVG) subunitpolypeptides which are modified specifically to alter a feature of thepolypeptide unrelated to its physiological activity. For example,cysteine residues can be substituted or deleted to prevent unwanteddisulfide linkages. Similarly, certain amino acids can be changed toenhance expression of a human N-type calcium channel hα_(1B+SFVG)subunit polypeptide by eliminating proteolysis by proteases in anexpression system (e.g., dibasic amino acid residues in yeast expressionsystems in which KEX2 protease activity is present).

Mutations of a nucleic acid which encode a human N-type calcium channelhα_(1B+SFVG) subunit polypeptide preferably preserve the amino acidreading frame of the coding sequence, and preferably do not createregions in the nucleic acid which are likely to hybridize to formsecondary structures, such as hairpins or loops, which can bedeleterious to expression of the variant polypeptide.

Mutations can be made by selecting an amino acid substitution, or byrandom mutagenesis of a selected site in a nucleic acid which encodesthe polypeptide. Variant polypeptides are then expressed and tested forone or more activities to determine which mutation provides a variantpolypeptide with a desired property. Further mutations can be made tovariants (or to non-variant human N-type calcium channel hα_(1B+SFVG)subunit polypeptides) which are silent as to the amino acid sequence ofthe polypeptide, but which provide preferred codons for translation in aparticular host. The preferred codons for translation of a nucleic acidin, e.g., E. coli, are well known to those of ordinary skill in the art.Still other mutations can be made to the noncoding sequences of a humanN-type calcium channel hα_(1B+SFVG) subunit gene or cDNA clone toenhance expression of the polypeptide.

The activity of variants of human N-type calcium channel hα_(1B+SFVG)subunit polypeptides can be tested by cloning the gene encoding thevariant human N-type calcium channel hα_(1B+SFVG) subunit polypeptideinto a bacterial or mammalian expression vector, introducing the vectorinto an appropriate host cell, expressing the variant human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide, and testing for afunctional capability of the human N-type calcium channel hα_(1B+SFVG)subunit polypeptides as disclosed herein. For example, the variant humanN-type calcium channel hα_(1B+SFVG) subunit polypeptide can be testedfor ability to provide voltage regulated calcium influx, as set forthbelow in the examples. Preparation of other variant polypeptides mayfavor testing of other activities, as will be known to one of ordinaryskill in the art.

The skilled artisan will also realize that conservative amino acidsubstitutions may be made in human N-type calcium channel hα_(1B+SFVG)subunit polypeptides to provide functionally equivalent variants of theforegoing polypeptides, i.e., variants which retain the functionalcapabilities of the human N-type calcium channel hα_(1B+SFVG) subunitpolypeptides. As used herein, a “conservative amino acid substitution”refers to an amino acid substitution which does not alter the relativecharge or size characteristics of the polypeptide in which the aminoacid substitution is made. Variants can be prepared according to methodsfor altering polypeptide sequence known to one of ordinary skill in theart such as are found in references which compile such methods, e.g.Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, etal., eds., John Wiley & Sons, Inc., New York. Exemplary functionallyequivalent variants of the human N-type calcium channel hα_(1B+SFVG)subunit polypeptides include conservative amino acid substitutions ofSEQ ID NO:4, but excluding the portion of the polypeptide consisting ofSEQ ID NO:2 (SFVG). Conservative substitutions of amino acids includesubstitutions made amongst amino acids within the following groups: (a)M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and(g) E, D.

Conservative amino-acid substitutions in the amino acid sequence ofhuman N-type calcium channel hα_(1B+SFVG) subunit polypeptide to producefunctionally equivalent variants of human N-type calcium channelhα_(1B+SFVG) subunit polypeptides typically are made by alteration ofthe nucleic acid sequence encoding human N-type calcium channelhα_(1B+SFVG) subunit polypeptides (e.g., SEQ ID NO:3). Suchsubstitutions can be made by a variety of methods known to one ofordinary skill in the art. For example, amino acid substitutions may bemade by PCR-directed mutation, site-directed mutagenesis according tothe method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488–492,1985), or by chemical synthesis of a gene encoding a human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide. Where amino acidsubstitutions are made to a small unique fragment of a human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide, such as a leucinezipper domain, the substitutions can be made by directly synthesizingthe peptide. The activity of functionally equivalent fragments of humanN-type calcium channel hα_(1B+SFVG) subunit polypeptides can be testedby cloning the gene encoding the altered human N-type calcium channelhα_(1B+SFVG) subunit polypeptide into a bacterial or mammalianexpression vector, introducing the vector into an appropriate host cell,expressing the altered human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide, and testing for the ability of the human N-type calciumchannel hα_(1B+SFVG) subunit polypeptide to transduce voltage regulatedcalcium influx. Peptides which are chemically synthesized can be testeddirectly for function.

A variety of methodologies well-known to the skilled practitioner can beutilized to obtain isolated human N-type calcium channel hα_(1B+SFVG)subunit molecules. The polypeptide may be purified from cells whichnaturally produce the polypeptide by chromatographic means orimmunological recognition. Alternatively, an expression vector may beintroduced into cells to cause production of the polypeptide. In anothermethod, mRNA transcripts may be microinjected or otherwise introducedinto cells to cause production of the encoded polypeptide. Translationof mRNA in cell-free extracts such as the reticulocyte lysate systemalso may be used to produce polypeptide. Those skilled in the art alsocan readily follow known methods for isolating human N-type calciumchannel hα_(1B+SFVG) subunit polypeptides. These include, but are notlimited to, immunochromatography, HPLC, size-exclusion chromatography,ion-exchange chromatography and immune-affinity chromatography.

The invention as described herein has a number of uses, some of whichare described elsewhere herein. For example, the invention permitsisolation of the human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide molecules containing the amino acid sequence of SEQ ID NO:2by e.g., expression of a recombinant nucleic acid to produce largequantities of polypeptide which may be isolated using standardprotocols. As another example, the isolation of the human N-type calciumchannel hα_(1B+SFVG) subunit gene makes it possible for the artisan todiagnose a disorder characterized by loss of expression of human N-typecalcium channel hα_(1B+SFVG) subunit. These methods involve determiningexpression of the human N-type calcium channel hα_(1B+SFVG) subunitnucleic acid, and/or human N-type calcium channel hα_(1B+SFVG) subunitpolypeptides derived therefrom. In the former situation, suchdeterminations can be carried out via any standard nucleic aciddetermination assay, including the polymerase chain reaction, orassaying with labeled hybridization probes.

The invention also embraces agents which bind selectively to the humanN-type calcium channel hα_(1B+SFVG) subunit (having or encoding SEQ IDNO:2) and agents which bind preferentially to the human N-type calciumchannel hα_(1B+SFVG) subunit (having or encoding SEQ ID NO:2) as well asagents which bind to variants and fragments of the polypeptides andnucleic acids as described herein. Selective binding means that theagent binds to the human N-type calcium channel hα_(1B+SFVG) subunit butnot to human N-type calcium channel non-hα_(1B+SFVG) subunits (i.e.,those subunits which do not have or encode SEQ ID NO:2). Preferentialbinding means that the agent binds more to the human N-type calciumchannel hα_(1B+SFVG) subunit than to human N-type calcium channelnon-hα_(1B+SFVG) subunit, e.g., the agent binds with greater affinity oravidity to the human N-type calcium channel hα_(1B+SFVG) subunit havingor encoding SEQ ID NO:2. The agents include polypeptides which bind tohuman N-type calcium channel hα_(1B+SFVG) subunit, and antisense nucleicacids, both of which are described in greater detail below. The agentscan inhibit or increase human N-type calcium channel hα_(1B+SFVG)subunit activity (antagonists and agonists, respectively).

Some of the agents are inhibitors. A human N-type calcium channelhα_(1B+SFVG) subunit inhibitor is an agent that inhibits human N-typecalcium channel hα_(1B+SFVG) subunit mediated voltage gated calciuminflux. Human N-type calcium channel hα_(1B+SFVG) subunit inhibitorsalso include dominant negative peptides and known N-type calcium channelinhibitors including the ω-conotoxin peptides and derivative thereofsuch as ziconotide (SNX-111). Small organic molecule calcium channelinhibitors, such as fluspirilene, NNC09-0026(−)-trans-1-butyl-4-(4-dimethylaminophenyl)-3-[(4-trifluoromethyl-phenoxy)methyl] piperidinedihydrochloride); SB 201823-A(4-[2-(3,4-dichlorophenoxy)ethyl]-1-pentyl piperidinehydrochloride); NS649 (2-amino-1-(2,5-dimethoxyphenyl)-5-trifluoromethyl benzimidazole);CNS 1237 (N-acenaphthyl-N′-4-methoxynaphth-1-yl guanidine) and riluzolemay also exhibit specificity for the human N-type calcium channelhα_(1B+SFVG) subunit.

Calcium influx assays can be performed to screen and/or determinewhether a human N-type calcium channel hα_(1B+SFVG) subunit inhibitorhas the ability to inhibit human N-type calcium channel hα_(1B+SFVG)subunit activity, and whether the inhibition is selective. As usedherein, “inhibit” refers to inhibiting by at least 10% voltage gatedcalcium influx, preferably inhibiting by at least 25% voltage gatedcalcium influx, and more preferably inhibiting by at least 40% voltagegated calcium influx as measured by any of the methods well known in theart. An exemplary assay of voltage gated calcium influx is describedbelow in the Examples.

Inhibitors may selectively inhibit hα_(1B+SFVG) based on the state ofdepolarization of the membrane with which the hα_(1B+SFVG) isassociated. It is well known that certain compounds preferentially bindto voltage-gated calcium channels at particular voltages. For example,dihydropyridine compounds preferentially bind to L-type voltage-gatedcalcium channels when the membrane is depolarized. Bean (Proc. Nat'l.Acad. Sci. 81:6388, 1984) described the binding of nitrendipine tocardiac L-type channels only when the membrane is depolarized. Similarresults have been found for nimodipine action in sensory neurons(McCarthy & TanPiengco, J. Neurosci. 12:2225, 1992).

Activators of human N-type calcium channel hα_(1B+SFVG) activity alsoare enhanced by the invention. Activators may be identified and/ortested using methods described above for inhibitors. The SFVG site islocated in a portion of the hα_(1B+SFVG) channel which is important forvoltage dependent gating of Ca²⁺ influx. Therefore, in screening formodulators of hα_(1B+SFVG), including inhibitors and activators (i.e.antagonists and agonists), it is preferred that compounds (e.g.libraries of potential channel inhibitors) are tested for modulation ofhα_(1B+SFVG) activity at a variety of voltages which cause partial orcomplete membrane depolarization, or hyperpolarization. These assays areconducted according to standard procedures of testing calcium channelfunction (e.g. patch clamping, fluorescent Ca²⁺ influx assays) whichrequire no more than routine experimentation. Using such methods,modulators of hα_(1B+SFVG) activity which are active at particularvoltages (e.g. complete membrane depolarization) can be identified. Suchcompounds are useful for selectively modulating calcium channel activityin conditions which may display voltage dependence. For example,following a stroke membranes are depolarized and such compounds may beactive in selectively blocking calcium channel activity for treatment ofstroke. Other uses will be apparent to one of ordinary skill in the art.

In one embodiment the human N-type calcium channel hα_(1B+SFVG) subunitinhibitor is an antisense oligonucleotide that selectively binds to ahuman N-type calcium channel hα_(1B+SFVG) subunit nucleic acid molecule,to reduce the expression of human N-type calcium channel hα_(1B+SFVG)subunit in a cell. This is desirable in virtually any medical conditionwherein a reduction of human N-type calcium channel hα_(1B+SFVG) subunitactivity is desirable, e.g., voltage gated calcium influx.

As used herein, the term “antisense oligonucleotide” or “antisense”describes an oligonucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridizes under physiological conditionsto DNA comprising a particular gene or to an mRNA transcript of thatgene and, thereby, inhibits the transcription of that gene and/or thetranslation of that mRNA. The antisense molecules are designed so as tointerfere with transcription or translation of a target gene uponhybridization with the target gene or transcript. Those skilled in theart will recognize that the exact length of the antisenseoligonucleotide and its degree of complementarity with its target willdepend upon the specific target selected, including the sequence of thetarget and the particular bases which comprise that sequence. It ispreferred that the antisense oligonucleotide be constructed and arrangedso as to bind selectively with the target under physiologicalconditions, i.e., to hybridize substantially more to the target sequencethan to any other sequence in the target cell under physiologicalconditions. Based upon SEQ ID NO:1, or upon allelic or homologousgenomic and/or cDNA sequences, one of skill in the art can easily chooseand synthesize any of a number of appropriate antisense molecules foruse in accordance with the present invention. In order to besufficiently selective and potent for inhibition, such antisenseoligonucleotides should comprise at least 10 and, more preferably, atleast 15 consecutive bases which are complementary to the target,although in certain cases modified oligonucleotides as short as 7 basesin length have been used successfully as antisense oligonucleotides(Wagner et al., Nature Biotechnol. 14:840–844, 1996). Most preferably,the antisense oligonucleotides comprise a complementary sequence of20–30 bases. Although oligonucleotides may be chosen which are antisenseto any region of the gene or mRNA transcripts, in preferred embodimentsthe antisense oligonucleotides correspond to N-terminal or 5′ upstreamsites such as translation initiation, transcription initiation orpromoter sites. In addition, 3′-untranslated regions may be targeted.Targeting to mRNA splicing sites has also been used in the art but maybe less preferred if alternative mRNA splicing occurs. In addition, theantisense is targeted, preferably, to sites in which mRNA secondarystructure is not expected (see, e.g., Sainio et al., Cell Mol.Neurobiol. 14(5):439–457, 1994) and at which polypeptides are notexpected to bind. Thus, the present invention also provides forantisense oligonucleotides which are complementary to allelic orhomologous cDNAs and genomic DNAs corresponding to human N-type calciumchannel hα_(1B+SFVG) subunit nucleic acid containing SEQ ID NO:1.

In one set of embodiments, the antisense oligonucleotides of theinvention may be composed of “natural” deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared by artrecognized methods which may be carried out manually or by an automatedsynthesizer. They also may be produced recombinantly by vectors.

In preferred embodiments, however, the antisense oligonucleotides of theinvention also may include “modified” oligonucleotides. That is, theoligonucleotides may be modified in a number of ways which do notprevent them from hybridizing to their target but which enhance theirstability or targeting or which otherwise enhance their therapeuticeffectiveness.

The term “modified oligonucleotide” as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic internucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5′ end of onenucleotide and the 3′ end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acids has been covalentlyattached to the oligonucleotide. Preferred synthetic internucleosidelinkages are phosphorothioates, alkylphosphonates, phosphorodithioates,phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates,carbonates, phosphate triesters, acetamidates, carboxymethyl esters andpeptides.

The term “modified oligonucleotide” also encompasses oligonucleotideswith a covalently modified base and/or sugar. For example, modifiedoligonucleotides include oligonucleotides having backbone sugars whichare covalently attached to low molecular weight organic groups otherthan a hydroxyl group at the 3′ position and other than a phosphategroup at the 5′ position. Thus modified oligonucleotides may include a2′-O-alkylated ribose group. In addition, modified oligonucleotides mayinclude sugars such as arabinose instead of ribose. The presentinvention, thus, contemplates pharmaceutical preparations containingmodified antisense molecules that are complementary to and hybridizablewith, under physiological conditions, nucleic acids encoding humanN-type calcium channel hα_(1B+SFVG) subunit polypeptides, together withpharmaceutically acceptable carriers.

Antisense oligonucleotides may be administered as part of apharmaceutical composition. Such a pharmaceutical composition mayinclude the antisense oligonucleotides in combination with any standardpharmaceutically acceptable carriers which are known in the art. Thecompositions should be sterile and contain a therapeutically effectiveamount of the antisense oligonucleotides in a unit of weight or volumesuitable for administration to a patient. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredients. Thecharacteristics of the carrier will depend on the route ofadministration. Pharmaceutically acceptable carriers include diluents,fillers, salts, buffers, stabilizers, solubilizers, and other materialswhich are well known in the art.

Agents which bind human N-type calcium channel hα_(1B+SFVG) subunit alsoinclude binding peptides which bind to the human N-type calcium channelhα_(1B+SFVG) subunit and complexes containing the human N-type calciumchannel hα_(1B+SFVG) subunit. When the binding polypeptides areinhibitors, the polypeptides bind to and inhibit the activity of humanN-type calcium channel hα_(1B+SFVG) subunit. To determine whether ahuman N-type calcium channel hα_(1B+SFVG) subunit binding peptide bindsto human N-type calcium channel hα_(1B+SFVG) subunit any known bindingassay may be employed. For example, the peptide may be immobilized on asurface and then contacted with a labeled human N-type calcium channelhα_(1B+SFVG) subunit. The amount of human N-type calcium channelhα_(1B+SFVG) subunit which interacts with the human N-type calciumchannel hα_(1B+SFVG) subunit binding peptide or the amount which doesnot bind to the human N-type calcium channel hα_(1B+SFVG) subunitbinding peptide may then be quantitated to determine whether the humanN-type calcium channel hα_(1B+SFVG) subunit binding peptide binds tohuman N-type calcium channel hα_(1B+SFVG) subunit. Further, the bindingof a human N-type calcium channel hα_(1B+SFVG) subunit and a humanN-type calcium channel non-hα_(1B+SFVG) subunit can be compared todetermine if the binding peptide binds selectively or preferentially.

The human N-type calcium channel hα_(1B+SFVG) subunit binding peptidesinclude peptides of numerous size and type that bind selectively orpreferentially to human N-type calcium channel hα_(1B+SFVG) subunitpolypeptides, and complexes of both human N-type calcium channelhα_(1B+SFVG) subunit polypeptides and their binding partners. Thesepeptides may be derived from a variety of sources. For example, bindingpeptides include known N-type calcium channel inhibitors such as theω-conotoxin peptides GVIA SEQ ID NO: 11 (from C. geographus) and MVIIASEQ ID NO: 12 (from C. magus). Other such human N-type calcium channelhα_(1B+SFVG) subunit binding peptides can be provided by modifying theforegoing peptides or by screening degenerate peptide libraries whichcan be readily prepared in solution, in immobilized form or as phagedisplay libraries. Combinatorial libraries also can be synthesized ofpeptides containing one or more amino acids. Libraries further can besynthesized of peptoids and non-peptide synthetic moieties.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent, for example, a completely degenerate orbiased array. One then can select phage-bearing inserts which bind tothe human N-type calcium channel hα_(1B+SFVG) subunit polypeptide. Thisprocess can be repeated through several cycles of reselection of phagethat bind to the human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide. Repeated rounds lead to enrichment of phage bearingparticular sequences. DNA sequence analysis can be conducted to identifythe sequences of the expressed polypeptides. The minimal linear portionof the sequence that binds to the human N-type calcium channelhα_(1B+SFVG) subunit polypeptide can be determined. One can repeat theprocedure using a biased library containing inserts containing part orall of the minimal linear portion plus one or more additional degenerateresidues upstream or downstream thereof. Yeast two-hybrid screeningmethods also may be used to identify polypeptides that bind to the humanN-type calcium channel hα_(1B+SFVG) subunit polypeptides. Thus, thehuman N-type calcium channel hα_(1B+SFVG) subunit polypeptides of theinvention, or a fragment thereof, can be used to screen peptidelibraries, including phage display libraries, to identify and selectpeptide binding partners of the human N-type calcium channelhα_(1B+SFVG) subunit polypeptides of the invention. Such molecules canbe used, as described, for screening assays, for purification protocols,for interfering directly with the functioning of human N-type calciumchannel hα_(1B+SFVG) subunit and for other purposes that will beapparent to those of ordinary skill in the art.

Peptides may easily be synthesized or produced by recombinant means bythose of skill in the art. Using routine procedures known to those ofordinary skill in the art, one can determine whether a peptide whichbinds to human N-type calcium channel hα_(1B+SFVG) subunit is usefulaccording to the invention by determining whether the peptide is onewhich inhibits the activity of human N-type calcium channel hα_(1B+SFVG)subunit in a voltage gated calcium influx assay, as discussed above.

The human N-type calcium channel hα_(1B+SFVG) subunit binding peptideagent may also be an antibody or a functionally active antibodyfragment. Antibodies are well known to those of ordinary skill in thescience of immunology. As used herein, the term “antibody” means notonly intact antibody molecules but also fragments of antibody moleculesretaining human N-type calcium channel hα_(1B+SFVG) subunit bindingability. Such fragments are also well known in the art and are regularlyemployed both in vitro and in vivo. In particular, as used herein, theterm “antibody” means not only intact immunoglobulin molecules but alsothe well-known active fragments F(ab′)₂, and Fab. F(ab′)₂, and Fabfragments which lack the Fc fragment of intact antibody, clear morerapidly from the circulation, and may have less non-specific tissuebinding of an intact antibody (Wahl et al., J. Nucl. Med. 24:316–325(1983)).

In one set of embodiments, the antibody useful according to the methodsof the present invention is an intact, fully human anti-human N-typecalcium channel hα_(1B+SFVG) subunit monoclonal antibody in an isolatedform or in a pharmaceutical preparation. The following is a descriptionof a method for developing a monoclonal antibody that interacts with andinhibits the activity of human N-type calcium channel hα_(1B+SFVG)subunit. The description is exemplary and is provided for illustrativepurposes only.

Murine monoclonal antibodies may be made by any of the methods known inthe art utilizing human N-type calcium channel hα_(1B+SFVG) subunit, ora fragment thereof, as an immunogen provided that the channel orfragment contains the amino acid sequence of SEQ ID NO:2.

Human monoclonal antibodies may be made by any of the methods known inthe art, such as those disclosed in U.S. Pat. No. 5,567,610, issued toBorrebaeck et al., U.S. Pat. No. 5,565,354, issued to Ostberg, U.S. Pat.No. 5,571,893, issued to Baker et al, Kozber, J. Immunol. 133: 3001(1984), Brodeur, et al., Monoclonal Antibody Production Techniques andApplications, p. 51–63 (Marcel Dekker, Inc, new York, 1987), and Boerneret al., J. Immunol., 147: 86–95 (1991). In addition to the conventionalmethods for preparing human monoclonal antibodies, such antibodies mayalso be prepared by immunizing transgenic animals that are capable ofproducing human antibodies (e.g., Jakobovits et al., Proc. Nat'l. Acad.Sci. USA, 90: 2551 (1993), Jakobovits et al., Nature, 362: 255–258(1993), Bruggermann et al., Year in Immuno., 7:33 (1993) and U.S. Pat.No. 5,569,825 issued to Lonberg).

Alternatively the antibody may be a polyclonal antibody specific forhuman N-type calcium channel hα_(1B+SFVG) subunit which inhibits humanN-type calcium channel hα_(1B+SFVG) subunit activity. The preparationand use of polyclonal antibodies is known to one of ordinary skill inthe art.

Significantly, as is well known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

In general, intact antibodies are said to contain “Fe” and “Fab”regions. The Fc regions are involved in complement activation and arenot involved in antigen binding. An antibody from which the Fc′ regionhas been enzymatically cleaved, or which has been produced without theFc′ region, designated an “F(ab′)₂” fragment, retains both of theantigen binding sites of the intact antibody. Similarly, an antibodyfrom which the Fc region has been enzymatically cleaved, or which hasbeen produced without the Fc region, designated an “Fab′” fragment,retains one of the antigen binding sites of the intact antibody. Fab′fragments consist of a covalently bound antibody light chain and aportion of the antibody heavy chain, denoted “Fd.” The Fd fragments arethe major determinants of antibody specificity (a single Fd fragment maybe associated with up to ten different light chains without alteringantibody specificity). Isolated Fd fragments retain the ability tospecifically bind to antigen epitopes.

The sequences of the antigen-binding Fab′ portion of the anti-humanN-type calcium channel hα_(1B+SFVG) subunit monoclonal antibodiesidentified as being useful according to the invention in the assaysprovided above, as well as the relevant FR and CDR regions, can bedetermined using amino acid sequencing methods that are routine in theart. It is well established that non-CDR regions of a mammalian antibodymay be replaced with corresponding regions of non-specific orhetero-specific antibodies while retaining the epitope specificity ofthe original antibody. This technique is useful for the development anduse of “humanized” antibodies in which non-human CDRs are covalentlyjoined to human FR and/or Fc/pFc′ regions to produce a functionalantibody. Techniques to humanize antibodies are particularly useful whennon-human animal (e.g., murine) antibodies which inhibit human N-typecalcium channel hα_(1B+SFVG) subunit activity are identified. Thesenon-human animal antibodies can be humanized for use in the treatment ofa human subject in the methods according to the invention. An example ofa method for humanizing a murine antibody is provided in PCTInternational Publication No. WO 92/04381 which teaches the productionand use of humanized murine RSV antibodies in which at least a portionof the murine FR regions have been replaced by FR regions of humanorigin. Such antibodies, including fragments of intact antibodies withantigen-binding ability, are often referred to as “chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, and Fab fragments of ananti-human N-type calcium channel hα_(1B+SFVG) subunit monoclonalantibody; chimeric antibodies in which the Fc and/or FR and/or CDR1and/or CDR2 and/or light chain CDR3 regions of an anti-human N-typecalcium channel hα_(1B+SFVG) subunit antibody have been replaced byhomologous human or non-human sequences; chimeric F(ab′)₂ fragmentantibodies in which the FR and/or CDR1 and/or CDR2 and/or light chainCDR3 regions of an anti-human N-type calcium channel hα_(1B+SFVG)subunit antibody have been replaced by homologous human or non-humansequences; and chimeric Fab fragment antibodies in which the FR and/orCDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced byhomologous human or non-human sequences. Thus, those skilled in the artmay alter an anti-human N-type calcium channel hα_(1B+SFVG) subunitantibody by the construction of CDR grafted or chimeric antibodies orantibody fragments containing, all or part thereof, of the disclosedheavy and light chain V-region CDR amino acid sequences (Jones et al.,Nature 321:522, 1986; Verhoeyen et al., Science 39:1534, 1988 andTempest et al., Bio/Technology 9:266, 1991), without destroying thespecificity of the antibodies for human N-type calcium channelhα_(1B+SFVG) subunit. Such CDR grafted or chimeric antibodies orantibody fragments can be effective in inhibiting human N-type calciumchannel hα_(1B+SFVG) subunit activity in animals (e.g. primates) andhumans.

In preferred embodiments, the chimeric antibodies of the invention arefully human monoclonal antibodies. As noted above, such chimericantibodies may be produced in which some or all of the FR regions ofhuman N-type calcium channel hα_(1B+SFVG) subunit have been replaced byother homologous human FR regions. In addition, the Fe portions may bereplaced so as to produce IgA or IgM as well as IgG antibodies bearingsome or all of the CDRs of the anti-human N-type calcium channelhα_(1B+SFVG) subunit antibody. Of particular importance is the inclusionof the anti-human N-type calcium channel hα_(1B+SFVG) subunit heavychain CDR3 region and, to a lesser extent, the other CDRs of anti-humanN-type calcium channel hα_(1B+SFVG) subunit antibodies. Such fully humanchimeric antibodies will have particular utility in that they will notevoke an immune response against the antibody itself.

It is also possible, in accordance with the present invention, toproduce chimeric antibodies including non-human sequences. Thus, one mayuse, for example, murine, ovine, equine, bovine or other mammalian Fc orFR sequences to replace some or all of the Fc or FR regions of theanti-human N-type calcium channel hα_(1B+SFVG) subunit antibody. Some ofthe CDRs may be replaced as well. Again, however, it is preferred thatat least the heavy chain CDR3 region of the anti-human N-type calciumchannel hα_(1B+SFVG) subunit antibody be included in such chimericantibodies and, to a lesser extent, it is also preferred that some orall of the other CDRs of anti-human N-type calcium channel hα_(1B+SFVG)subunit be included. Such chimeric antibodies bearing non-humanimmunoglobulin sequences admixed with the CDRs of the human anti-humanN-type calcium channel hα_(1B+SFVG) subunit monoclonal antibody are notpreferred for use in humans and are particularly not preferred forextended use because they may evoke an immune response against thenon-human sequences. They may, of course, be used for brief periods orin immunosuppressed individuals but, again, fully human antibodies arepreferred. Because, however, such antibodies may be used for briefperiods or in immunosuppressed subjects, chimeric antibodies bearingnon-human mammalian Fc and FR sequences but including at least theanti-human N-type calcium channel hα_(1B+SFVG) subunit heavy chain CDR3are contemplated as alternative embodiments of the present invention.

For inoculation or prophylactic uses, the antibodies of the presentinvention are preferably intact antibody molecules including the Fcregion. Such intact antibodies will have longer half-lives than smallerfragment antibodies (e.g. Fab) and are more suitable for intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal administration.

Fab fragments, including chimeric Fab fragments, are preferred inmethods in which the antibodies of the invention are administereddirectly to a local tissue environment. For example, the Fab fragmentsare preferred when the antibody of the invention is administereddirectly to the brain. Fabs offer several advantages over F(ab′)₂ andwhole immunoglobulin molecules for this therapeutic modality. First,because Fabs have only one binding site for their cognate antigen, theformation of immune complexes is precluded whereas such complexes can begenerated when bivalent F(ab′)₂ s and whole immunoglobulin moleculesencounter their target antigen. This is of some importance becauseimmune complex deposition in tissues can produce adverse inflammatoryreactions. Second, because Fabs lack an Fc region they cannot triggeradverse inflammatory reactions that are activated by Fc, such asactivation of the complement cascade. Third, the tissue penetration ofthe small Fab molecule is likely to be much better than that of thelarger whole antibody. Fourth, Fabs can be produced easily andinexpensively in bacteria, such as E. coli, whereas whole immunoglobulinantibody molecules require mammalian cells for their production inuseful amounts. Production of Fabs in E. coli makes it possible toproduce these antibody fragments in large fermenters which are lessexpensive than cell culture-derived products.

Smaller antibody fragments and small binding peptides having bindingspecificity for the human N-type calcium channel hα_(1B+SFVG) subunitwhich can be used to inhibit human N-type calcium channel hα_(1B+SFVG)subunit activity also are embraced within the present invention. Forexample, single-chain antibodies can be constructed in accordance withthe methods described in U.S. Pat. No. 4,946,778 to Ladner et al. Suchsingle-chain antibodies include the variable regions of the light andheavy chains joined by a flexible linker moiety. Methods for obtaining asingle domain antibody (“Fd”) which comprises an isolated VH singledomain, also have been reported (see, for example, Ward et al., Nature341:644–646 (1989)).

According to the invention human N-type calcium channel hα_(1B+SFVG)subunit inhibitors also include “dominant negative” polypeptides derivedfrom SEQ ID NO:4. A dominant negative polypeptide is an inactive variantof a polypeptide, which, by interacting with the cellular machinery,displaces an active polypeptide from its interaction with the cellularmachinery or competes with the active polypeptide, thereby reducing theeffect of the active polypeptide. For example, a dominant negativereceptor which binds a ligand but does not transmit a signal in responseto binding of the ligand can reduce the biological effect of expressionof the ligand. Likewise, a dominant negative human N-type calciumchannel hα_(1B+SFVG) subunit of an active complex (e.g. N-type calciumchannel) can interact with the complex but prevent the activity of thecomplex (e.g. voltage gated calcium influx).

The end result of the expression of a dominant negative human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide of the invention in acell is a reduction in voltage gated calcium influx. One of ordinaryskill in the art can assess the potential for a dominant negativevariant of a human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide, and using standard mutagenesis techniques to create one ormore dominant negative variant polypeptides. For example, given theteachings contained herein of a human N-type calcium channelhα_(1B+SFVG) subunit polypeptide, one of ordinary skill in the art canmodify the sequence of the human N-type calcium channel hα_(1+SFVG)subunit polypeptide by site-specific mutagenesis, scanning mutagenesis,partial gene deletion or truncation, and the like. See, e.g., U.S. Pat.No. 5,580,723 and Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Theskilled artisan then can test the population of mutagenized polypeptidesfor diminution in human N-type calcium channel hα_(1B+SFVG) subunitactivity (e.g., voltage gated calcium influx) and/or for retention ofsuch an activity. Other similar methods for creating and testingdominant negative variants of a human N-type calcium channelhα_(1B+SFVG) subunit polypeptide will be apparent to one of ordinaryskill in the art.

Each of the compositions of the invention is useful for a variety oftherapeutic and non-therapeutic purposes. For example, the human N-typecalcium channel hα_(1B+SFVG) subunit nucleic acids of the invention areuseful as oligonucleotide probes. Such oligonucleotide probes can beused herein to identify genomic or cDNA library clones possessing anidentical or substantially similar nucleic acid sequence. A suitableoligonucleotide or set of oligonucleotides, which is capable ofhybridizing under stringent hybridization conditions to the desiredsequence, a variant or fragment thereof, or an anti-sense complement ofsuch an oligonucleotide or set of oligonucleotides, can be synthesizedby means well known in the art (see, for example, Synthesis andApplication of DNA and RNA, S. A. Narang, ed., 1987, Academic Press, SanDiego, Calif.) and employed as a probe to identify and isolate thedesired sequence, variant or fragment thereof by techniques known in theart. Techniques of nucleic acid hybridization and clone identificationare disclosed by Sambrook, et al., Molecular Cloning, A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.(1989), and by Hames, B. D., et al., in Nucleic Acid Hybridization, APractical Approach, IRL Press, Washington, D.C. (1985). To facilitatethe detection of a desired nucleic acid sequence, or variant or fragmentthereof, whether for cloning purposes or for the mere detection of thepresence of the sequence, the above-described probes may be labeled witha detectable group. Such a detectable group may be any material having adetectable physical or chemical property. Such materials have beenwell-developed in the field of nucleic acid hybridization and, ingeneral, most any label useful in such methods can be applied to thepresent invention. Particularly useful are radioactive labels. Anyradioactive label may be employed which provides for an adequate signaland has a sufficient half-life. If single stranded, the oligonucleotidemay be radioactively labeled using kinase reactions. Alternatively,oligonucleotides are also useful as nucleic acid hybridization probeswhen labeled with a non-radioactive marker such as biotin, an enzyme ora fluorescent group. See, for example, Leary, J. J., et al., Proc. Natl.Acad. Sci. (USA) 80:4045 (1983); Renz, M. et al., Nucl. Acids Res.12:3435 (1984); and Renz, M., EMBO J. 6:817 (1983).

Additionally, complements of the human N-type calcium channelhα_(1B+SFVG) subunit nucleic acids can be useful as antisenseoligonucleotides, e.g., by delivering the antisense oligonucleotide toan animal to induce a human N-type calcium channel hα_(1B+SFVG) subunit“knockout” phenotype. The administration of antisense RNA probes toblock gene expression is discussed in Lichtenstein, C., Nature333:801–802 (1988).

Alternatively, the human N-type calcium channel hα_(1B+SFVG) subunitnucleic acid of the invention can be used to prepare a non-humantransgenic animal. A “transgenic animal” is an animal having cells thatcontain DNA which has been artificially inserted into a cell, which DNAbecomes part of the genome of the animal which develops from that cell.Preferred transgenic animals are primates, mice, rats, cows, pigs,horses, goats, sheep, dogs and cats. Animals suitable for transgenicexperiments can be obtained from standard commercial sources such asCharles River (Wilmington, Mass.), Taconic (Germantown, N.Y.), HarlanSprague Dawley (Indianapolis, Ind.), etc. Transgenic animals having aparticular property associated with a particular disease can be used tostudy the affects of a variety of drugs and treatment methods on thedisease, and thus serve as genetic models for the study of a number ofhuman diseases. The invention, therefore, contemplates the use of humanN-type calcium channel hα_(1B+SFVG) subunit knockout and transgenicanimals as models for the study of disorders involving voltage gatedcalcium influx.

A variety of methods are available for the production of transgenicanimals associated with this invention. DNA can be injected into thepronucleus of a fertilized egg before fusion of the male and femalepronuclei, or injected into the nucleus of an embryonic cell (e.g., thenucleus of a two-cell embryo) following the initiation of cell division.See e.g., Brinster et al., Proc. Nat. Acad. Sci. USA, 82: 4438 (1985);Brinster et al., Cell 27: 223 (1981); Costantini et al., Nature 294: 982(1981); Harpers et al., Nature 293: 540 (1981); Wagner et al., Proc.Nat. Acad. Sci. USA 78:5016 (1981); Gordon et al., Proc. Nat. Acad. Sci.USA 73: 1260 (1976). The fertilized egg is then implanted into theuterus of the recipient female and allowed to develop into an animal.

An alternative method for producing transgenic animals involves theincorporation of the desired gene sequence into a virus which is capableof affecting the cells of a host animal. See e.g., Elbrecht et al.,Molec. Cell. Biol. 7: 1276 (1987); Lacey et al., Nature 322: 609 (1986);Leopol et al., Cell 51: 885 (1987). Embryos can be infected withviruses, especially retroviruses, modified to carry the nucleotidesequences of the invention which encode human N-type calcium channelhα_(1B+SFVG) subunit proteins or sequences which disrupt the nativehuman N-type calcium channel hα_(1B+SFVG) subunit gene to produce aknockout animal.

Another method for producing transgenic animals involves the injectionof pluripotent embryonic stem cells into a blastocyst of a developingembryo. Pluripotent stem cells derived from the inner cell mass of theembryo and stabilized in culture can be manipulated in culture toincorporate nucleotide sequences of the invention. A transgenic animalcan be produced from such cells through implantation into a blastocystthat is implanted into a foster mother and allowed to come to term. Seee.g., Robertson et al., Cold Spring Harbor Conference Cell Proliferation10: 647 (1983); Bradley et al., Nature 309: 255 (1984); Wagner et al.,Cold Spring Harbor Symposium Quantitative Biology 50: 691 (1985).

The procedures for manipulation of the rodent embryo and formicroinjection of DNA into the pronucleus of the zygote are well knownto those of ordinary skill in the art (Hogan et al., supra).Microinjection procedures for fish, amphibian eggs and birds aredetailed in Houdebine and Chourrout, Experientia, 47: 897–905 (1991).Other procedures for introduction of DNA into tissues of animals aredescribed in U.S. Pat. No., 4,945,050 (Sandford et al., Jul. 30, 1990).

By way of example only, to prepare a transgenic mouse, female mice areinduced to superovulate. Females are placed with males, and the matedfemales are sacrificed by CO₂ asphyxiation or cervical dislocation andembryos are recovered from excised oviducts. Surrounding cumulus cellsare removed. Pronuclear embryos are then washed and stored until thetime of injection. Randomly cycling adult female mice are paired withvasectomized males. Recipient females are mated at the same time asdonor females. Embryos then are transferred surgically. The procedurefor generating transgenic rats is similar to that of mice. See Hammer etal., Cell, 63:1099–1112 (1990).

Methods for the culturing of embryonic stem (ES) cells and thesubsequent production of transgenic animals by the introduction of DNAinto ES cells using methods such as etectroporation, calciumphosphate/DNA precipitation and direct injection also are well known tothose of ordinary skill in the art. See, for example, Teratocarcinomasand Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed.,IRL Press (1987).

In cases involving random gene integration, a clone containing thesequence(s) of the invention is co-transfected with a gene encodingresistance. Alternatively, the gene encoding neomycin resistance isphysically linked to the sequence(s) of the invention. Transfection andisolation of desired clones are carried out by any one of severalmethods well known to those of ordinary skill in the art (E. J.Robertson, supra).

DNA molecules introduced into ES cells can also be integrated into thechromosome through the process of homologous recombination. Capecchi,Science, 244: 1288–1292 (1989). Methods for positive selection of therecombination event (e.g., neo resistance) and dual positive-negativeselection (e.g., neo resistance and gancyclovir resistance) and thesubsequent identification of the desired clones by PCR have beendescribed by Capecchi, supra and Joyner et al., Nature, 338: 153–156(1989). The final phase of the procedure is to inject targeted ES cellsinto blastocysts and to transfer the blastocysts into pseudopregnantfemales. The resulting chimeric animals are bred and the offspring areanalyzed by Southern blotting to identify individuals that carry thetransgene.

Procedures for the production of non-rodent mammals and other animalshave been discussed by others. See Houdebine and Chourrout, supra;Pursel et al., Science 244: 1281–1288 (1989); and Simms et al.,Bio/Technology, 6: 179–183 (1988).

Inactivation or replacement of the endogenous N-type calcium channelhα_(1B+SFVG) subunit gene can be achieved by a homologous recombinationsystem using embryonic stem cells. The resultant transgenic non-humanmammals (preferably primates) having a knockout N-type calcium channelhα_(1B+SFVG) subunit characteristic may be made transgenic for the humanN-type calcium channel hα_(1B+SFVG) subunit and used as a model forscreening compounds as modulators (agonists or antagonists/inhibitors)of the human N-type calcium channel hα_(1B+SFVG) subunit. In thismanner, such therapeutic drugs can be identified.

Additionally, a normal or mutant version of human N-type calcium channelhα_(1B+SFVG) subunit can be inserted into the mouse (or the animal) germline to produce transgenic animals which constitutively or induciblyexpress the normal or mutant form of human N-type calcium channelhα_(1B+SFVG) subunit. These animals are useful in studies to define therole and function of human N-type calcium channel hα_(1B+SFVG) subunitin cells. These studies are particularly useful in animals, which do notnormally express human N-type calcium channel hα_(1B+SFVG) subunit, suchas non-primates.

A human N-type calcium channel hα_(1B+SFVG) subunit polypeptide, or afragment thereof, also can be used to isolate human N-type calciumchannel hα_(1B+SFVG) subunit native binding partners, including, e.g.,the N-type calcium channel. Isolation of such binding partners may beperformed according to well-known methods. For example, isolated humanN-type calcium channel hα_(1B+SFVG) subunit polypeptides can be attachedto a substrate (e.g., chromatographic media, such as polystyrene beads,or a filter), and then a solution suspected of containing the N-typecalcium channel may be applied to the substrate. If a N-type calciumchannel which can interact with human N-type calcium channelhα_(1B+SFVG) subunit polypeptides is present in the solution, then itwill bind to the substrate-bound human N-type calcium channelhα_(1B+SFVG) subunit polypeptide. The N-type calcium channel then may beisolated. Other polypeptides which are binding partners for human N-typecalcium channel hα_(1B+SFVG) subunit may be isolated by similar methodswithout undue experimentation.

The compositions of the invention are also useful for therapeuticpurposes. Accordingly the invention encompasses a method for inhibitinghuman N-type calcium channel hα_(1B+SFVG) subunit activity in amammalian cell. The invention further provides methods for reducing orincreasing human N-type calcium channel hα_(1B+SFVG) subunit activity ina cell. In one embodiment, the method involves contacting the mammaliancell with an amount of a human N-type calcium channel hα_(1B+SFVG)subunit inhibitor effective to inhibit voltage gated calcium influx inthe mammalian cell. Such methods are useful in vitro for alteringvoltage gated calcium influx for the purpose of, for example,elucidating the mechanisms involved in stroke, pain, e.g., neuropathicpain, and traumatic brain injury and for restoring the voltage gatedcalcium influx in a cell having a defective human N-type calcium channelhα_(1B+SFVG) subunit. In vivo, such methods are useful, for example, forreducing N-type voltage gated calcium influx, e.g., to treat stroke,pain, e.g., neuropathic pain, traumatic brain injury, or any conditionin which human N-type calcium channel hα_(1B+SFVG) subunit activity iselevated.

An amount of a human N-type calcium channel hα_(1B+SFVG) subunitinhibitor which is effective to inhibit voltage gated calcium influx inthe mammalian cell is an amount which is sufficient to reduce voltagegated calcium influx by at least 10%, preferably at least 20%, morepreferably 30% and still more preferably 40%. An amount of a humanN-type calcium channel hα_(1B+SFVG) subunit which is effective toincrease voltage gated calcium influx in the mammalian cell is an amountwhich is sufficient to increase voltage gated calcium influx by at least10%, preferably at least 20%, more preferably 30% and still morepreferably 40%. Such alterations in voltage gated calcium influx can bemeasured by the assays described herein.

As described above with respect to inhibitors, modulators ofhα_(1B+SFVG) may selectively inhibit or increase hα_(1B+SFVG) functionbased on the state of depolarization of the membrane with which thehα_(1B+SFVG) is associated. Therefore, in screening for modulators ofhα_(1B+SFVG) it is preferred that compounds (e.g. syntheticcombinatorial libraries, natural products, peptide libraries, etc.) aretested for modulation of hα_(1B+SFVG) activity at a variety of voltageswhich cause partial or complete membrane depolarization, orhyperpolarization. These assays are conducted according to standardprocedures of testing calcium channel function (e.g. patch clamping,fluorescent Ca²⁺ influx assays) which require no more than routineexperimentation. Using such methods, modulators of hα_(1B+SFVG) activitywhich are active at particular voltages (e.g. complete membranedepolarization) can be identified. Such compounds are useful forselectively modulating calcium channel activity in conditions which maydisplay voltage dependence.

The invention also encompasses a method for increasing human N-typecalcium channel hα_(1B+SFVG) subunit expression in a cell or subject. Itis desirable to increase human N-type calcium channel hα_(1B+SFVG)subunit in a subject that has a disorder characterized by a deficiencyin voltage gated calcium influx. The amount of human N-type calciumchannel hα_(1B+SFVG) subunit can be increased in such cell or subject bycontacting the cell with, or administering to the subject, a humanN-type calcium channel hα_(1B+SFVG) subunit nucleic acid or a humanN-type calcium channel hα_(1B+SFVG) subunit polypeptide of the inventionto the subject in an amount effective to increase voltage gated calciuminflux in the cell or the subject. An increase in human N-type calciumchannel hα_(1B+SFVG) subunit activity can be measured by the assaysdescribed herein, e.g., assays of calcium influx.

The invention also contemplates gene therapy. The procedure forperforming ex vivo gene therapy is outlined in U.S. Pat. No. 5,399,346and in exhibits submitted in the file history of that patent, all ofwhich are publicly available documents. In general, it involvesintroduction in vitro of a functional copy of a gene into a cell(s) of asubject which contains a defective copy of the gene, and returning thegenetically engineered cell(s) to the subject. The functional copy ofthe gene is under operable control of regulatory elements which permitexpression of the gene in the genetically engineered cell(s). Numeroustransfection and transduction techniques as well as appropriateexpression vectors are well known to those of ordinary skill in the art,some of which are described in PCT application WO95/00654. In vivo genetherapy using vectors such as adenovirus, retroviruses, herpes virus,and targeted liposomes also is contemplated according to the invention.

The preparations of the invention are administered in effective amounts.An effective amount is that amount of a pharmaceutical preparation thatalone, or together with further doses, produces the desired response. Inthe case of treating a condition characterized by aberrant voltage gatedcalcium influx, the desired response is reducing or increasing calciuminflux to a level which is within a normal range. Preferably, the changein calcium influx produces a detectable reduction in a physiologicalfunction related to the condition, e.g., a reduction in neurotoxicityfollowing stroke. The responses can be monitored by routine methods. Inthe case of a condition where an increase in voltage gated calciuminflux is desired, an effective amount is that amount necessary toincrease said influx in the target tissue. The converse is the case whena reduction in influx is desired. An increase or decrease inneurotransmitter release also could be measured to monitor the response.

Such amounts will depend, of course, on the particular condition beingtreated, the severity of the condition, the individual patientparameters including age, physical condition, size and weight, theduration of the treatment, the nature of concurrent therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practitioner. It is preferredgenerally that a maximum dose be used, that is, the highest safe doseaccording to sound medical judgment. It will be understood by those ofordinary skill in the art, however, that a patient may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reasons.

Generally, doses of active compounds would be from about 0.01 mg/kg perday to 1000 mg/kg per day. It is expected that doses ranging from 50–500mg/kg will be suitable and in one or several administrations per day.Lower doses will result from other forms of administration, such asintravenous administration. In the event that a response in a subject isinsufficient at the initial doses applied, higher doses (or effectivelyhigher doses by a different, more localized delivery route) may beemployed to the extent that patient tolerance permits. Multiple dosesper day are contemplated to achieve appropriate systemic levels ofcompound, although fewer doses typically will be given when compoundsare prepared as slow release or sustained release medications.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptably compositions. Such preparations mayroutinely contain salts, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic agents. When used inmedicine, the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically-acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

The human N-type calcium channel hα_(1B+SFVG) subunit inhibitors orhuman N-type calcium channel hα_(1B+SFVG) subunit nucleic acids andpolypeptides useful according to the invention may be combined,optionally, with a pharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; and phosphoricacid in a salt.

The pharmaceutical compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular compound selected,the severity of the condition being treated and the dosage required fortherapeutic efficacy. The methods of the invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, topical,nasal, interdermal, or parenteral routes. The term “parenteral” includessubcutaneous, intravenous, intrathecal, intramuscular, or infusion.Intravenous or intramuscular routes are not particularly suitable forlong-term therapy and prophylaxis.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the human N-type calciumchannel hα_(1B+SFVG) subunit inhibitor or human N-type calcium channelhα_(1B+SFVG) subunit nucleic acids and polypeptides, which is preferablyisotonic with the blood of the recipient. This aqueous preparation maybe formulated according to known methods using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationalso may be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1,3-butane diol. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution, and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono-or di-glycerides. In addition,fatty acids such as oleic acid may be used in the preparation ofinjectables. Carrier formulation suitable for oral, subcutaneous,intravenous, intrathecal, intramuscular, etc. administrations can befound in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa.

Other delivery systems can include time-release, delayed release orsustained release delivery systems such as the biological/chemicalvectors is discussed above. Such systems can avoid repeatedadministrations of the active compound, increasing convenience to thesubject and the physician. Many types of release delivery systems areavailable and known to those of ordinary skill in the art. Use of along-term sustained release implant may be desirable. Long-term release,are used herein, means that the implant is constructed and arranged todelivery therapeutic levels of the active ingredient for at least 30days, and preferably 60 days. Long-term sustained release implants arewell-known to those of ordinary skill in the art and include some of therelease systems described above.

The invention further provides efficient methods of identifyingpharmacological agents or lead compounds for agents useful in thetreatment of conditions associated with aberrant voltage gated cellcalcium influx mediated by human N-type calcium channel hα_(1B+SFVG)subunit and the compounds and agents so identified. Generally, thescreening methods involve assaying for compounds which inhibit orenhance voltage gated calcium influx through human N-type calciumchannels. Such methods are adaptable to automated, high throughputscreening of compounds. Examples of such methods are described in U.S.Pat. No. 5,429,921.

A variety of assays for pharmacological agents are provided, including,labeled in vitro protein binding assays, Ca²⁺influx assays, etc. Forexample, protein binding screens are used to rapidly examine the bindingof candidate pharmacological agents to a human N-type calcium channelhα_(1B+SFVG) subunit. The candidate pharmacological agents can bederived from, for example, combinatorial peptide libraries. Convenientreagents for such assays are known in the art. An exemplary cell-basedassay of calcium influx involves contacting a neuronal cell having ahuman N-type calcium channel hα_(1B+SFVG) subunit with a candidatepharmacological agent under conditions whereby the influx of calcium canbe stimulated by application of a voltage to the test system, i.e., bymembrane depolarization. Specific conditions are well known in the artand are described in Lin et al., Neuron 18:153–166, 1997, and in U.S.Pat. No. 5,429,921. A reduction in the voltage gated calcium influx inthe presence of the candidate pharmacological agent indicates that thecandidate pharmacological agent reduces the induction of calcium influxof human N-type calcium channel hα_(1B+SFVG) subunit in response to thevoltage stimulus. An increase in the voltage gated calcium influx in thepresence of the candidate pharmacological agent indicates that thecandidate pharmacological agent increases the induction of calciuminflux of human N-type calcium channel hα_(1B+SFVG) subunit in responseto the voltage stimulus. Methods for determining changes in theintracellular calcium concentration are known in the art and areaddressed elsewhere herein.

Human N-type calcium channel hα_(1B+SFVG) subunit used in the methods ofthe invention can be added to an assay mixture as an isolatedpolypeptide (where binding of a candidate pharmaceutical agent is to bemeasured) or as a cell or other membrane-encapsulated space whichincludes a human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide. In the latter assay configuration, the cell or othermembrane-encapsulated space can contain the human N-type calcium channelhα_(1B+SFVG) subunit as a preloaded polypeptide or as a nucleic acid(e.g. a cell transfected with an expression vector containing a humanN-type calcium channel hα_(1B+SFVG) subunit). In the assays describedherein, the human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide can be produced recombinantly, or isolated from biologicalextracts, but preferably is synthesized in vitro. Human N-type calciumchannel hα_(1B+SFVG) subunit polypeptides encompass chimeric proteinscomprising a fusion of a human N-type calcium channel hα_(1B+SFVG)subunit polypeptide with another polypeptide, e.g., a polypeptidecapable of providing or enhancing protein-protein binding, or enhancingstability of the human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide under assay conditions. A polypeptide fused to a humanN-type calcium channel hα_(1B+SFVG) subunit polypeptide or fragmentthereof may also provide means of readily detecting the fusion protein,e.g., by immunological recognition or by fluorescent labeling.

The assay mixture also comprises a candidate pharmacological agent.Typically, a plurality of assay mixtures are run in parallel withdifferent agent concentrations to obtain a different response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e., at zero concentration of agent or at aconcentration of agent below the limits of assay detection. Candidateagents encompass numerous chemical classes, although typically they areorganic compounds. Preferably, the candidate pharmacological agents aresmall organic compounds, i.e., those having a molecular weight of morethan 50 yet less than about 2500. Candidate agents comprise functionalchemical groups necessary for structural interactions with polypeptides,and typically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups andmore preferably at least three of the functional chemical groups. Thecandidate agents can comprise cyclic carbon or heterocyclic structureand/or aromatic or polyaromatic structures substituted with one or moreof the above-identified functional groups. Candidate agents also can bebiomolecules such as peptides, saccharides, fatty acids, sterols,isoprenoids, purines, pyrimidines, derivatives or structural analogs ofthe above, or combinations thereof and the like. Where the agent is anucleic acid, the agent typically is a DNA or RNA molecule, althoughmodified nucleic acids having non-natural bonds or subunits are alsocontemplated.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides, synthetic organic combinatorial libraries, phagedisplay libraries of random peptides, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bereadily modified through conventional chemical, physical, andbiochemical means. Further, known pharmacological agents may besubjected to directed or random chemical modifications such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs of the agents.

Candidate agents can be selected randomly or can be based on existingcompounds which bind to and/or modulate the function of N-type calciumchannels. For example, compounds which are known to inhibit N-typecalcium channels include fluspirilene, ziconotide (SNX-111), theω-conotoxin peptides GVIA (SEQ ID NO: 11) and MVIIA (SEQ ID NO: 12), aswell as small organic molecule calcium channel inhibitors, such asfluspirilene, NNC09-0026(−)-trans-1-butyl-4-(4-dimethylaminophenyl)-3-[(4-trifluoromethyl-phenoxy)methyl]piperidinedihydrochloride); SB 201823-A(4-[2-(3,4-dichlorophenoxy)ethyl]-1-pentyl piperidinehydrochloride); NS649 (2-amino-1-(2,5-dimethoxyphenyl)-5-trifluoromethyl benzimidazole);CNS 1237 (N-acenaphthyl-N′-4-methoxynaphth-1-yl guanidine) and riluzole.Therefore, a source of candidate agents are libraries of molecules basedon the foregoing N-type calcium channel inhibitors, in which thestructure of the inhibitor is changed at one or more positions of themolecule to contain more or fewer chemical moieties or differentchemical moieties. The structural changes made to the molecules increating the libraries of analog inhibitors can be directed, random, ora combination of both directed and random substitutions and/oradditions. One of ordinary skill in the art in the preparation ofcombinatorial libraries can readily prepare such libraries based on theexisting N-type calcium channel inhibitors.

A variety of other reagents also can be included in the mixture. Theseinclude reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc. which may be used to facilitate optimalprotein-protein and/or protein-nucleic acid binding. Such a reagent mayalso reduce non-specific or background interactions of the reactioncomponents. Other reagents that improve the efficiency of the assay suchas protease inhibitors, nuclease inhibitors, antimicrobial agents, andthe like may also be used.

The mixture of the foregoing assay materials is incubated underconditions whereby, but for the presence of the candidatepharmacological agent, the human N-type calcium channel hα_(1B+SFVG)subunit transduces a control amount of voltage gated calcium influx. Fordetermining the binding of a candidate pharmaceutical agent to a humanN-type calcium channel hα_(1B+SFVG) subunit, the mixture is incubatedunder conditions which permit binding. The order of addition ofcomponents, incubation temperature, time of incubation, and otherparameters of the assay may be readily determined. Such experimentationmerely involves optimization of the assay parameters, not thefundamental composition of the assay. Incubation temperatures typicallyare between 4° C. and 40° C. Incubation times preferably are minimizedto facilitate rapid, high throughput screening, and typically arebetween 1 minute and 10 hours.

After incubation, the level of voltage gated calcium influx or the levelof specific binding between the human N-type calcium channelhα_(1B+SFVG) subunit polypeptide and the candidate pharmaceutical agentis detected by any convenient method available to the user. For cellfree binding type assays, a separation step is often used to separatebound from unbound components. The separation step may be accomplishedin a variety of ways. Conveniently, at least one of the components isimmobilized on a solid substrate, from which the unbound components maybe easily separated. The solid substrate can be made of a wide varietyof materials and in a wide variety of shapes, e.g., microtiter plate,microbead, dipstick, resin particle, etc. The substrate preferably ischosen to maximize signal to noise ratios, primarily to minimizebackground binding, as well as for ease of separation and cost.

Separation may be effected for example, by removing a bead or dipstickfrom a reservoir, emptying or diluting a reservoir such as a microtiterplate well, rinsing a bead, particle, chromatographic column or filterwith a wash solution or solvent. The separation step preferably includesmultiple rinses or washes. For example, when the solid substrate is amicrotiter plate, the wells may be washed several times with a washingsolution, which typically includes those components of the incubationmixture that do not participate in specific bindings such as salts,buffer, detergent, non-specific protein, etc. Where the solid substrateis a magnetic bead, the beads may be washed one or more times with awashing solution and isolated using a magnet.

Detection may be effected in any convenient way for cell-based assayssuch as a calcium influx assay. The calcium influx resulting fromvoltage stimulus of the human N-type calcium channel hα_(1B+SFVG)subunit polypeptide typically alters a directly or indirectly detectableproduct, e.g., a calcium sensitive molecule such as fura-2-AM. For cellfree binding assays, one of the components usually comprises, or iscoupled to, a detectable label. A wide variety of labels can be used,such as those that provide direct detection (e.g., radioactivity,luminescence, optical or electron density, etc). or indirect detection(e.g., epitope tag such as the FLAG epitope, enzyme tag such ashorseradish peroxidase, etc.). The label may be bound to a human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide or the candidatepharmacological agent.

A variety of methods may be used to detect the label, depending on thenature of the label and other assay components. For example, the labelmay be detected while bound to the solid substrate or subsequent toseparation from the solid substrate. Labels may be directly detectedthrough optical or electron density, radioactive emissions, nonradiativeenergy transfers, etc. or indirectly detected with antibody conjugates,streptavidin-biotin conjugates, etc. Methods for detecting the labelsare well known in the art.

The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention.

EXAMPLES Example 1 Analysis of Human Brain N-type Calcium Channel SpliceVariants

The abundance of splice variants of N-type calcium channels in humanbrain was determined using polymerase chain reaction analysis and RNaseprotection assays as described in Lin et al. (Neuron 18:153–166, 1997).Human N-type calcium channel α_(1B) subunit clones were sequenced bystandard methods of nucleotide sequencing and it was determined that onetype of clone had a 12 nucleotide insert (SEQ ID NO:1) as compared topreviously published human N-type calcium channel α_(1B) subunitsequences. The present human N-type calcium channel α_(1B) subunitnucleic acid molecule (designated hα_(1B+SFVG)) corresponds to thepublished nucleotide sequence for human N-type calcium channel α_(1B)subunits with the 12 nucleotide insert located after nucleotide 3855 (asnumbered in Williams et al., Science 257:389–395, 1992). The nucleotidesequence of SEQ ID NO:1 supplies the third base of the codon encodingSer1237, three new codons (Ser1238, Phe1239 and Val1240), and the firsttwo bases of codon Gly1241, as shown in SEQ ID NO:3: tcG AGC TTC GTG GGa(insert in caps). This insert thus encodes a four amino acid insert inthe protein which is similar to, but surprisingly is not identical to,amino acids 1236–1239 of a rat N-type calcium channel α_(1B) subunit(SEQ ID NO:10, GenBank accession number M92905). The human N-typecalcium channel hα_(1B+SFVG) subunit was found to make up a significantportion of the N-type calcium channel α_(1B) subunits mRNA in humanbrain. It was also determined that the hα_(1B+SFVG) subunit wasdifferentially distributed in different parts of the brain, e.g. incertain portions of the brain hα_(1B+SFVG) was more highly expressedthan in other portions of the brain.

Example 2 Construction of Human N-type Calcium Channel hα_(1B+SFVG)Subunit Nucleic Acids

The human N-type calcium channel hα_(1B+SFVG) subunit containing theSFVG insert is constructed according to standard procedures describedin, e.g., Current Protocols in Molecular Biology (F. M. Ausubel, et al.,eds., John Wiley & Sons, Inc., New York), using PCR primers whichcontain the nucleotides encoding SFVG (e.g., SEQ ID NO:1) to amplify thepublished human N-type calcium channel α_(1B) subunit nucleic acid.Fragments generated by PCR are then assembled by ligation to prepare acomplete cDNA encoding the human hα_(1B+SFVG) subunit.

Example 3 Function of the Human N-type Calcium Channel hα_(1B+SFVG)Subunit

The voltage gated calcium channel activity of the human N-type calciumchannel hα_(1B+SFVG) subunit is tested using to the methods described inLin et al. (1997) for a rat N-type calcium channel subunit, and asdescribed in Example 4 below.

Example 4 Activation Differences in Rat N-type Calcium Channels ±the ETExon

Functional Assessment of the Calcium Channel α_(1B) cDNA Constructs

The functional properties of all calcium (Ca) channel α_(1B) cDNAconstructs described in this paper were assessed in the Xenopus oocyteexpression system. All methods and procedures were essentially the sameas described in Lin et al. (1997). cRNAs were in vitro transcribed usingthe mMESSAGE mMACHINE kit (Ambion) from the various α_(1B) cDNAconstructs subcloned into the Xenopus β-globin expression vector (pBSTA;Goldin & Sumikawa et al., Methods Enzymol 207:279–297, 1992). 46 nl of a750 ng/μl cRNA solution was injected into defolliculated oocytes using aprecision nanoinjector (Drummond). N-type Ca channel currents wererecorded 6–7 days after injection. At least 15 minutes prior torecording, oocytes were injected with 46 nl of a 50 mM solution of BAPTA(1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetate). This we havefound critical to minimize activation of an endogenous Ca²⁺-activatedCl⁻current, even when Ba²⁺is the charge carrier (Lin et al., 1997).Cells exhibiting slowly deactivating tail currents, indicative of thepresence of Ba²⁺-dependent activation of the Ca-activated Cl⁻ current,were excluded from the analysis.

N-type Ca²⁺channel currents were recorded from oocytes using the twomicroelectrode voltage-clamp recording technique (Warner amplifier;OC-725b). A virtual ground circuit eliminated the need for seriesresistance compensation when recording large currents. Micropipettes of0.8–1.5 MΩ and 0.3–0.5MΩ resistance when filled with 3 M KCl were usedfor the voltage and current recording electrodes, respectively. Oocytesexpressing Ca²⁺channel currents usually had resting membrane potentialsbetween −40 and −50 mV when impaled with two electrodes. A groundedmetal shield was placed between the two electrodes to increase thesettling time of the clamp. Recording solutions contained 5 mM BaCl₂, 85mM tetraethylammonium, 5 mM KCl, and 5 mM HEPES (pH adjusted to 7.4 withmethanesulfonic acid). The recording temperature was between 19° C. and22° C.

The properties of each mutant construct were assessed by expressing ittogether with appropriate controls (ΔET α_(1B) and +ET α_(1B)). Eachmutant was tested in three separate batches of oocytes and within eachbatch, recordings were made from at least six oocytes for each mutantconstruct and control. Recordings from the oocytes expressing thevarious Ca channel Δ_(1B) constructs were randomized throughout the datacollection period.

Data Analysis

Data were acquired on-line and leak subtracted using a P/4 protocol(PClamp V6.0; Axon Inst.). Voltage-steps were applied every 10–30seconds depending on the duration of the step, from a holding potentialof −80 mV. Ca channel currents recorded under these conditions showedlittle run-down over the duration of the recordings. Three sets ofcurrent voltage-relationships were obtained from each cell using stepdepolarizations of 26.3 ms, 650 ms and 2.6 s in duration and digitizedat 25 kHz, 10 kHz and 250 Hz, respectively. Exponential curves(activation and inactivation) were fit to the data using curve fittingroutines in PClamp (Axon Instr.) and Origin (Microcal). Inactivationtime constants in the range of 70–800 msec were estimated from currentsevoked by the longest depolarization (2.6 s). Activation time constantswere best resolved from currents evoked by the shortest depolarizations(26.3 ms; sampled at 25 kHz).

Modeling Ca Entry.

A one-compartment cell model employing standard compartmental modelingtechniques in NEURON (Hines & Carnevale, Neural Comput. 9:1179–1209,1997) was used to predict the amount of Ca entering a neuron expressingeither rnα_(1B-b) or rnα_(1B-b) N-type Ca channel currents. The cell hada total membrane area of 1250 μm², 0.75 μF/cm² specific membranecapacitance and 30 kΩcm² specific membrane resistance. For actionpotential simulation a fast sodium conductance (g_(Na)) and a delayedrectifying potassium conductance (g_(K,DR)) were included (Mainen &Sejnowski, Science 268:1503–1506, 1995) each with densities of 300pS/μm². Ca²⁺influx was mediated by a fast calcium conductance (g_(Ca);Yamada et al., Multiple channels and calcium dynamics. In Methods inNeuronal Modeling, Koch, C. & Segev, I., Eds. pp 97–134, 1989) with adensity of 1 pS/μm². Resultant currents were calculated usingconventional Hodgkin-Huxley kinetic schemes according to the formulaegiven below. The resting membrane potential was set at −70 mV and Na andK current reversal potentials at +50 mV and −75 mV, respectively. Thecalcium channel was computed using the Goldman-Hodgkin-Katz equation.Extracellular Ca concentration was 2.5 mM and the intracellular Caconcentration computed using entry via I_(Ca) and removal via a firstorder pump d[Ca²⁺]_(i)/dt=(−1×10⁵.I_(Ca)/2F)−([Ca²⁺]₁−[Ca²⁺]_(∞))/τ_(R),where

-   [Ca²⁺]_(∞)=10 nM and σ_(R)=80 ms. The time constants and maximal    conductances were developed at room temperature and were therefore    scaled to 37° C. using a Q₁₀ of 2.3. Formulae used for calculation    of various currents were as follows:-   Sodium current (I_(na)), m³·h: α_(m),    _(Na)=0.182·(v+25)/(1−e^((v+25)/9)); β_(m),    _(Na)=−0.124·(v+25)/(1−e^(−(v+25)/9)) α_(h),    _(Na)=0.024·(v+40)/(1−e^(−(v+40)/5)); β_(h),    _(Na)=−0.0091·(v+65)/(1−e^(−(v+65)/5)); h_(∞),    _(Na)=1/(1−e^(−(v+55/62))-   Delayed rectifier (I_(K(DR))), m: α_(m),    _(K(DR))=0.02·(v−25)/(1−e^(−(v+25)/9)); βm,    _(K(DR))=−0.002·(v−25)/(1−e^((v+25)/9))-   High threshold, N-type calcium current (I_(ca)), m·h: m_(∞),    _(Ca)=1/(1+e^(−(v−3)/8)); τ_(m), _(Ca)=7.8/(e^((v+6)/16));    h_(Ca)=K/(K+[Ca²⁺]_(i)) with K=0.01 mM.

The brain-dominant form, rnα_(1B-d), was then modeled by shifting thevoltage-dependence of the N-type Ca channel conductance activationvariable (m_(∞), _(Ca)) by −7 mV, and decreasing the activation timeconstant (τ_(m), _(Ca)) by 33% (Lin et al., 1997 and see FIG. 1A).

Ribonuclease Protection Assay

The procedures are essentially the same as those described in Lin et al.(1997). Total RNA was purified from various neuronal tissue of adultrats using a guanidium thiocyanate and phenol-chloroform extractionprotocol (adapted from Chomczynski & Sacchi, Anal. Biochem. 162:156–159,1987). ³²P-labeled antisense RNA probes overlapping ET (nt 4379–4836) inrnα_(1B-b) and NP (nt4605–4930) in rbα_(1A) (Starr et al., Proc. Natl.Acad. Sci. USA, 88:5621–5625, 1991) were constructed from linearizedplasmids (pGEM-T vector) containing appropriate RT-PCR-derivedsub-clones using the Maxi-script kit (Ambion). Probes were gel purifiedand stored as ethanol precipitates. 1 μg of RNA purified fromsympathetic or sensory ganglia or 5 μg of RNA isolated from various CNStissues were precipitated with 2×10⁵ cpm of probe and resuspended in 30μl hybridization buffer containing: 60% formamide; 0.4 M NaCl; 10 mMEDTA and 40 mM PIPES at pH 6.4. Samples were denatured at 85° C. andallowed to hybridize overnight at 60° C. The samples were then digestedin a 350 μl reaction mix containing: 0.3 M NaCl, 5mM EDTA, 3.5 μl of theRNase Cocktail (Ambion) and 10 mM Tris at pH 7.5, then treated withproteinase K, extracted and precipitated with 10 μg of tRNA as carrier.After resuspension in 30 μl formamide loading buffer, the samples weedenatured and separated on a 5% polyacrylamide gel. After exposure to aphosphor imaging plate to quantity relative band intensities (Fuji BAS1000), the gel was subsequently exposed to film with an intensifyingscreen for 4–5 days at −80° C.

Site-directed Mutagenesis

A recombinant PCR-based technique was used to introduce mutations (QT,EA, AT, AA, NP) at the ET site in the IVS3-S4 linker of α_(1B-b). A pairof primers 5′-attcttgtggtcatcgccttgag (Bup 3460; SEQ ID NO: 13) and5′-gacaggcctccaggagcttggtg (Bdw 5623; SEQ ID NO: 14) flanked a region ofthe clone that contained two restriction sites RsrII (nt3510) and BglII(nt5465) located on either side of ET (nt4674). A second primer paircontained the desired mutation and directly overlapped the ET site(Bdwmut and Bupmut; see below). Two separate PCRs were performed withBup 3460 and Bdwmut, and Bupmut and Bdw 5623. The PCR product thenserved as template for a second round of PCR using Bup 3460 and Bdw 5623generating the final mutant PCR fragment that was subsequently subclonedinto rnα_(1B-b) at the Rsr II and Bgl II sites. Mutants were screened byrestriction digest and confirmed by DNA sequencing. All PCR wasperformed using Expand High Fidelity (Boehringer Mannheim). Themutagenesis primers used were as follows:

ET/AT: Bupmut 5′-gagattgcgGCAACGaacaacttcatc-3′: SEQ ID NO: 15 Bdwmut5′-aagttgttCGTTTCcgcaatctccg-3′; SEQ ID NO: 16 ET/QT: Bupmut5′-gagattgcgCAGACGaacaacttcatc-3′; SEQ ID NO: 17 Bdwmut5′-aagttgttCGTCTGcgcaatctccg-3′; SEQ ID NO: 18 ET/EA: Bupmut5′-gagattgcgGAAGCTaacaacttcatc-3′; SEQ ID NO: 19 Bdwmut5′-aagttgttAGCTTCcgcaatctccg-3′; SEQ ID NO: 20 ET/AA: Bupmut5′-gagattgcgGCAGCTaacaacttcatc-3′; SEQ ID NO: 21 Bdwmut5′-aagttgttAGCTGCcgcaatctccg-3′; SEQ ID NO: 22 ET/NP: Bupmut5′-gagattgcgAACCCTaacaacttcatc-3′; SEQ ID NO: 23 Bdwmut5′-aagttgttAGGGTTcgcaatctccg-3′; SEQ ID NO: 24Genomic Analysis

The IVS3-S4 region of the rat α_(1B) and α_(1A) genes were analyzed bygenomic PCR. Primer pairs were directed to the IVS3 and IVS4 membranespanning regions that were presumed to reside in the 5′ and 3′ exonsflanking the ET and NP insertions of the α_(1B) and α_(1A) genes,respectively. PCR was performed in a 50 μl reaction mix containing 250ng rat liver genomic DNA, 250 μM of each nucleotide and 0.4 μM of eachprimer. After a pre-incubation for 15 min at 92° C., 0.75 μl enzyme mixwas added to start the amplification. The resultant gDNA products weregel purified, cloned into pGEM-T (Promega) and sequenced. The α_(1B)primers generated two bands of ˜11 kb and ˜900 bases. The 11 kb band wasderived from the α_(1B) gene and contained the desired ET encoding exonin IVS3-S4. The 900 base product resulted from amplification of theequivalent site in the α_(1E) gene that contained a relatively short˜700 bp intron and no intervening exon. The α_(1A) primers generated asingle 9 kb PCR product that was confirmed to be derived from the α_(1A)gene by DNA sequencing (Yale University sequencing facility). Primerswere as follows:

α_(1A): Aup4737 5′-tgcctggaacatcttcgactttgtga; SEQ ID NO: 25 Adw48765′-cagaggagaatgcggatggtgtaacc; SEQ ID NO: 26 α_(1B): Bup45995′-cagagatgcctggaacgtctttgac; SEQ ID NO: 27 Bdw47445′-ataacaagatgcggatggtgtagcc; SEQ ID NO: 28Alternative Splicing in the Putative S3-S4 Extracellular Linkers AffectsChannel Activation but Not Inactivation Kinetics

In a previous study it was shown that rnα_(1B-b) (ΔSFMG/+ET) andrnα_(1B-b) (+SFMG/ΔET) N-type currents differ with respect to theiractivation kinetics when expressed in Xenopus oocytes (compare Δ/+ and+/Δ in FIG. 1A,B; see also Lin et al., 1997). Inactivation kinetics ofthe two splice variants have not, however, been compared (Lin et al.,1997). In the present study depolarizations of durations of between 26ms and 2.6 s were employed to permit the resolution of both the timecourse of Ca channel activation and inactivation. Rat N-type calciumchannel subunits (rnα_(1B-b) [Δ/+] and rnα_(1B-d) [+/Δ]) were expressedin Xenopus oocytes and resulting N-type Ca channel currents recordedusing 5 mM Ba as the charge carrier (FIG. 1). FIG. 1A shows theaveraged, normalized Ca channel current induced by the expression inXenopus oocytes of four different α_(1B) constructs. Currents wereevoked by step depolarizations to 0 mV from a holding potential of −80mV. Each trace represents the average, normalized current calculatedfrom at least 6 oocytes. SFMG-containing clones are distinguished fromSFMG-lacking clones by thin and thick lines and arrows, respectively.FIG. 1B shows a plot of average activation time constants (nat. log) atdifferent test potentials (between −20 and +10 mV) for clones +/+ (□),Δ/+ (•), +/Δ (◯) and Δ/Δ (▪). The presence of SFMG in domain IIIS3-S4did not affect the rate of channel activation. There was no significantdifference in τ_(activ) between clones +/+ and Δ/+ or between clones +/Δand Δ/Δ (p>0.1 at all potentials between −20 mV and +10 mV). Thepresence of ET in domain IVS3-S4 slowed channel activation kinetics.τ_(activ) values for clones +/+ and Δ/+ were significantly slowercompared to +/Δ and Δ/Δ, at all test potentials between −20 mV and +10mV (p<0.05).

N-type Ca channel currents evoked by depolarization to 0 mV or higher,inactivated with a bi-exponential time course (τ_(fast) 100–150 ms andτ_(slow) 700–800 ms). The inactivation time constants of the clonedchannels expressed in Xenopus oocytes (rnα_(1B-b), Δ/+ and rnα_(1B-d),+/Δ) were weakly voltage-dependent consistent with studies of nativeN-type Ca channels of bullfrog sympathetic neurons (Jones & Marks,1989). The fast and slow inactivation time constants of rnα_(1B-b) andrnα_(1B-d) currents evoked by step depolarizations to between 0 mV and+mV were not significantly different. In contrast, the rates of channelactivation of the two variants in the same cells were significantlydifferent (FIG. 1 A,B). On the basis of these observations it wasconcluded that alternative splicing in domains IIIS3-S4 and IVS3-S4 ofthe α_(1B)-subunit altered the time course of N-type Ca channelactivation but had no effect on inactivation kinetics. These findingsare consistent with the close proximity of the S3-S4 linkers to theirrespective S4 helices that are the putative voltage sensors of the 6transmembrane family of voltage-gated ion channels. In contrast, thedomains of the Ca channels α_(1B) subunit implicated involtage-dependent inactivation of N-type Ca channels (IS6 and flankingputative extracellular and intracellular linkers; Zhang et al., Nature372:97–100, 1994) are likely to be more distant from the S3-S4 linkersplice sites.

The Observed Differences in the Properties of rnα_(1B-b) and rnα_(1B-d)Currents are of Sufficient Magnitude to Impact Action Potential-inducedCa Entry.

An assumption that motivates the present study is that the differencesin the kinetics and voltage-dependence of activation of rnα_(1B-b) andrnα_(1B-d) N-type Ca channel currents are sufficient to influence themagnitude and time course of voltage-dependent calcium entry in nativecells. A direct test of this hypothesis, however, is complicated by theinability to manipulate selectively the expression or activity ofindividual splice variants in their native environment. To date noisoform-specific pharmacological tools or antibodies to target Cachannel α_(1B) S3-S4 splice variants exist. Therefore, the availableinformation was used to estimate the relative effectiveness ofrnα_(1B-b) and rnα_(1B-d) N-type currents to support actionpotential-induced Ca influx in a model neuron (Hines & Carnevale, 1997).A one-compartment model was used to predict the time course andmagnitude of calcium entry in a neuron during action potential-induceddepolarization. Simulated action potentials with time courses similar tothose recorded in native sympathetic neurons (Yamada et al., 1989; FIG.2A) were used to trigger voltage-dependent Ca influx in model neurons(Na, K and Ca current densities of 300, 300 and 1 pS/μF, respectively)expressing either rnα_(1B-b) or rnα_(1B-d) N-type Ca channel currents. Asimulated action potential was evoked by a 10 ms, 40 pA current step(FIG. 2A); a comparison of the resultant N-type channel current (FIG.2B) and time course of intracellular calcium concentration (FIG. 2C)expected in a model neuron expressing either rnα_(1B-b) (Δ/+; solidline) or rnα_(1B-d) (+/Δ; dashed line)-type channels is shown. A shiftin the voltage-dependence of the N-type Ca channel conductanceactivation variable (m_(∞), _(Ca)) by −7 mV, and a decrease in theactivation time constrant (τ_(m, Ca)) by 33% expected for rnα_(1B-d)(Lin et al., 1997; and see FIG. 1A), resulted in a total increase incharge transfer and peak intracellular Ca concentration of 49% and 48%,respectively. A −50% increase in the total charge transfer (FIG. 2B) andpeak intracellular Ca concentration (FIG. 2C) is predicted during anaction potential in a neuron expressing rnα_(1B-d)-type Ca channels(dashed line) relative to rnα_(1B-b) (solid line). All other factorsbeing constant, the functional differences between rnα_(1B-b) andrnα_(1B-d) N-type Ca channel currents would be expected to significantlyimpact the amount of calcium that enters a neuron during actionpotential-dependent excitation.

Splicing of ET in Domain IVS3-S4 Underlies the Major FunctionalDifference Between rnα_(1B-b) and rnα_(1B-d)

rnα_(1B-b) and rnα_(1B-d) differ in composition by 6 amino acids locatedin two distinct regions of the Ca channel α_(1B) subunit (SFMG in domainIIIS3-S4 and ET in domain IVS3-S4). To separate the relativecontribution of SFMG in domain IIIS3-S4 and ET in domain IVS3-S4 to thedifferent gating kinetics observed between rnα_(1B-b) (ΔSFMG/+ET) andrnα_(1B-d) (+SFMG/ΔET) two additional clones, +/+ and Δ/Δ wereconstructed and the functional properties of all four clones werecompared. FIG. 1 (A and B) demonstrates that the presence of thedipeptide sequence ET in domain IVS3-S4 is directly correlated with thealtered activation kinetics of rnα_(1B-b) currents compared tornα_(1B-d). Activation time constants measured from N-type Ca channelcurrents in oocytes expressing clone Δ/+ (rnα_(1B-b)) and +/+ wereindistinguishable and 1.5 fold slower on average than those induced bythe expression of clones +/Δ (rnα_(1B-d)) and Δ/Δ (FIG. 1A,B). Thepresence of ET in domain IVS3-S4 also influenced the voltage-dependenceof channel activation. A comparison of the mid-points of the risingphase of the peak current-voltage plots (V_(1/2)) generated for the twoET containing clones, Δ/+ (rnα_(1B); −7.8±0.6 mV, n=6) and +/+ (−9.7±1.0mV, n=6) shows that they are not significantly different from each other(p>0.05, students' t-test). Likewise, V_(1/2) values estimated from twoET-lacking constructs, +/Δ (rnα_(1B-d); −15.4±0.4 mV, n=7) and Δ/Δ(−13.4±0.7, n=6), were not significantly different from each other(p>0.05) and activated at potentials that were, on average, 6 mV morenegative compared to ET-containing clones Δ/+ and +/+. While thepresence of ET in domain IVS3-S4 dominates in regulating thevoltage-dependence of activation, the analysis does reveal a smallcontribution of SFMG. SFMG-containing clones (+/Δ and +/+) activated atpotentials that were 2 mV hyperpolarized compared to those that lackedSFMG (Δ/+ and Δ/Δ). A 2 mV shift in the voltage-dependence of activationwas not significant at the 5% level, in a comparison of V_(1/2) valuesfrom clones Δ/+ and +/+, but did reach significance in a comparison of+/Δ and Δ/Δ (p<0.025, students t-test).

The Pattern of Expression of ET-containing Ca Channel α_(1B) mRNA inDifferent Regions of the Nervous System

FIG. 1 indicates that alternative splicing of ET within domain IVS3-S4of the Ca channel α_(1B)-subunit accounts for the major functionaldifferences between rnα_(1B-b) and rnα_(1B-A). This prompted a systemicanalysis of the expression pattern of the six bases in α_(1B) mRNA thatencoded ET (gaa acg). It was previously shown that ET-containing α_(1B)(+ET α_(1B)) mRNA was in very low abundance in total rat brain extracts(Lin et al., 1997). To determine whether ET-lacking α_(1B) (ΔET α_(1B))mRNA dominated throughout the central nervous system RNA isolated fromspinal cord, cerebellum, cortex, hippocampus, hypothalamus, medulla andthalamus of adult rats was analyzed by ribonuclease protection assay. Inall regions tested >90% of the α_(1B) mRNA expressed in the centralnervous system lacked the ET encoding sequence. In contrast, insympathetic and sensory ganglia the majority of α_(1B) mRNA containedthe ET encoding sequence. Together these findings suggest that +ETα_(1B) subunits are primarily restricted to neurons of the peripheralnervous system. Consistent with this RNA isolated from human brain andtrigeminal ganglia was analyzed and analogous patterns of expressionwere observed: low levels of +ET α_(1B) mRNA in brain and high levels(>90%) in ganglia.

Site-directed Mutagenesis within IVS3-S4

Having shown that alternative splicing of the ET encoding sequence inthe IVS3-S4 linker of α_(1B) has a significant effect on the kineticsand voltage-dependence of N-type Ca channel gating, the use ofsite-directed mutagenesis was employed to determine the relativeimportance of each amino acid, glutamate and threonine. A series ofmutants in which ET was replaced with either QT, AT, EA, AA or NP wereconstructed (FIG. 3) from clone Δ/+ (rnα_(1B-b)) which served as thebackground structure. The mutant constructs were then expressed inXenopus oocytes and their properties compared to clones +ET (100% slow;FIG. 3) and ΔET (100% fast; FIG. 3). All mutants expressed equally wellin the Xenopus oocyte expression system.

The role of the glutamate in domain IVS3-S4 was of major interestbecause it should be negatively charged at neutral pH and consequentlymight influence the gating machinery of the channel via electrostaticinteractions. FIG. 3, however, shows that replacing glutamate withglutamine resulted in a channel that activated only slightly faster than+ET α_(1B) (FIG. 3; QT). Substituting alanine for glutamate (AT)decreased τ_(act) but, consistent with the QT mutant, suggests that thepresence of a negative charge in IVS3-S4 (glu) does not underlie theslow gating kinetics of the +ET α_(1B) variant. Similarly, alaninesubstitution of either threonine alone (EA) or together with glutamate(AA) generated channels with activation kinetics that were intermediatebetween +ET α_(1B) and ΔET α_(1B) clones. Together, these resultssuggest that the presence of both glutamate and threonine in the IVS3-S4linker is necessary to reconstitute the relatively slow channel openingrates characteristic of N-type Ca channel α_(1B)-subunits that dominatein sensory and sympathetic ganglia.

Sequence comparisons of several cDNAs encoding α₁-subunits of othervoltage-gated Ca channels suggests that alternative splicing in theIVS3-S4 linker could be a general mechanism for regulatingvoltage-dependent Ca channel gating. This has recently been demonstratedfor α_(1A) (Sutton et al., Soc. Neurosci. Abs. 24:21, 1998), a Cachannel subunit that is closely related both structurally andfunctionally to the N-type Ca channel α_(1B) subunit. A comparison ofthe IVS3-S4 region of various mammalian α_(1A) cDNAs derived fromkidney, pancreas and brain (see also Yu et al., Proc. Natl. Acad. Sci.USA 89:10494–10498, 1992; Ligon et al., J Biol. Chem. 273:13905–13911,1998; Sutton et al., 1998) is consistent with alternative splicing ofsix bases encoding Asp Pro (NP) amino acids in this region. Thedistribution of +NP α_(1A) and ΔNP α_(1A) mRNAs in different regions ofthe rat nervous system has not been quantified. Therefore RNaseprotection analysis was used to determine the expression pattern of theIVS3-S4 splice variants of α_(1A). Low levels of +NP α_(1A) mRNA werefound in rat, spinal cord, striatum and thalamus, a pattern thatparallels the low levels of +ET α_(1B) mRNA in the CNS. However, thepattern of NP expression in the cerebellum, cortex and hippocampus didnot conform to this picture since mRNA isolated from these tissuescontained a significant proportion of +NP α_(1A) mRNAs. In fact, in thehippocampus +NP α_(1A) mRNAs dominated (˜60%). Consistent with theabundance of +ET α_(1B) mRNAs in peripheral tissue, the majority ofα_(1A) mRNA in superior cervical and dorsal root ganglia contained thesix bases encoding NP in domain IVS3-S4 of α_(1A). The absolute level ofα_(1A) mRNA expressed in sympathetic neurons was very low as expectedfrom the absence of P-type currents in recordings from rat sympatheticneurons (Mintz et al., 1992).

The high degree of sequence homology between α_(1B) and α_(1A) in theIVS3-S4 linker region together with the finding that a 6 base sequenceis alternatively spliced at both these sites, suggested that ET and NPshare a common functional role. To test this hypothesis the functionalimpact on N-type Ca channel currents of replacing ET in rnα_(1B-b) withNP was studied. FIG. 3 shows that the +NP α_(1B) mutant gives rise toN-type Ca channel currents in oocytes with gating kineticsindistinguishable from wild-type (i.e. +ET α_(1B). )Activation timeconstants were estimated from currents induced by the expression of thevarious mutant α_(1B) constructs (QT, AT, EA, AA, NP) in oocytes andcompared to clones ET and ΔET (A). Shifts in the activation timeconstants of the mutant channels, relative to clones ET and ΔET (100%slow) and ΔET (100% fast) are plotted (B). Each point represents datacollected from at least 18 oocytes per mutant (each mutant was tested inthree separate batches of oocytes and within each experiment at least 6oocytes per mutant were analyzed). Values plotted are means ± standarderrors from the three data sets. The asterisk indicates a significantslowing of the activation time constant compared to clone ET (P<0.05).

ET is Encoded By a Six Base Exon in the IVS3-S4 Linker Region of theα_(1B) Gene

The existence of an alternatively spliced exon in the IVS3-S4 region ofthe rat Ca channel α_(1B) gene has been hypothesized (Lin et al., 1997),but not yet confirmed. Genomic analysis was therefore undertaken tolocate the splice junctions in the IVS3-S4 region of the α_(1B) gene andto pinpoint the precise location of the putative six-base, ET encodingexon. PCR amplification from rat genomic DNA using primers designed tohybridize to the transmembrane spanning S3 and S4 helices flankingIVS3-S4 in α_(1B) revealed the presence of a long ˜10 kb stretch ofintron sequence. DNA sequencing established the location of exon/intronand intron/exon boundaries and conserved ag-gt splice junction signaturesequences immediately 5′ and 3′ to the putative ET insertion site. Asix-base cassette exon encoding ET was located 8 kb into the 5′ intronand establishes that ET-α_(1B) variants are generated by alternativesplicing. The exon/intron structure in the IVS3-S4 linker region of theclosely related rat α_(1A) gene was also determined. The rat α_(1A) genealso contained a long stretch of intron sequence (˜8 kb) and ag-gtsplice junctions at the 5′ (gt) and 3′ (at) ends of the intronicsegment. The precise location of the NP encoding cassette exon in therat α_(1A) gene has not been determined but conclude that it must residewithin the 8 kb of intron sequence in the IVS3-S4 linker region.Tissue-specific alternative splicing of six base cassette exons in theIVS3-S4 linkers of both α_(1A) and α_(1B) explains the presence ofsplice variants of these subunits in the mammalian brain and underscoresthe high level of conservation between these two functionally relatedgenes. The genomic structure of the more distantly related rat α_(1E)gene that encodes a pharmacologically and functionally distinct class ofCa channel (Soong et al., Science 260:1133–1136, 1993) also wasanalyzed. The α_(1E) gene contains a ˜700 bp intron in the IVS3-S4linker region and no obvious intervening exon. The absence of analternatively spliced cassette exon in the IVS3-S4 linker region of theα_(1E) gene is consistent with RNase protection analysis of α_(1E) mRNAfrom rat brain which revealed no evidence of sequence variations in thisIVS3-S4 linker.

Each of the foregoing patents, patent applications and references ishereby incorporated by reference. While the invention has been describedwith respect to certain embodiments, it should be appreciated that manymodifications and changes may be made by those of ordinary skill in theart without departing from the spirit of the invention. It is intendedthat such modification, changes and equivalents fall within the scope ofthe following claims.

1. An isolated human N-type calcium channel hα_(1B+SFVG) subunit nucleicacid molecule which encodes a human N-type calcium channel hα_(1B+SFVG)subunit polypeptide comprising SEQ ID NO:4.
 2. An isolated human N-typecalcium channel hα_(1B+SFVG) subunit nucleic acid molecule comprisingthe nucleotide sequence of SEQ ID NO:3.
 3. An isolated human N-typecalcium channel hα_(1B+SFVG) subunit nucleic acid molecule consisting ofthe nucleotide sequence of SEQ ID NO:3.
 4. An isolated fragment of thehuman N-type calcium channel hα_(1B+SFVG) subunit nucleic acid moleculeof claim 1, wherein the fragment comprises SEQ ID NO:1.
 5. An expressionvector comprising the isolated human N-type calcium channel hα_(1B+SFVG)subunit nucleic acid molecule of claim 1 operably linked to a promoter.6. An expression vector comprising the isolated fragment of the humanN-type calcium channel hα_(1B+SFVG) subunit nucleic acid molecule ofclaim 4 operably linked to a promoter.
 7. A host cell transformed ortransfectcd with the expression vector of claim 5.